Defending champion Dinara Safina, recovered from a back injury, lost her opener against Alexandra Dulgheru of Romania 6-4, 6-7 (5), 6-1.
Top-ranked Serena defeated Timea Bacsinszky 7-6 (2), 6-1 in her first match since winning the Australian Open more than three months ago. No. 4-ranked Venus, taking to the center court at the Foro Italico just before her sister, handled veteran Patty Schnyder 6-2, 6-2.
Serena overcame a slow start to come back and win the match. She was down 4-1 and saved three set points at 5-4 before going to a tiebreaker against Bacsinszky. After dominating the breaker, Serena reeled off the next four games to secure the match.
Serena said she didn't quite know what to expect in her first match since January.
"I went in there thinking I can win or I can lose. You never know what can happen and I didn't put too much pressure on myself," Serena said, adding that she felt good and was happy to pull off a win after a tentative beginning.
"I was a little off. I got off to a little bit of a slow start but it was good and I was able to fight it off," the 28-year-old American said. "It's a start."
Venus had an easier time against Schnyder, a lefty who had defeated Serena in Rome last year. Venus was always in control of the match, breaking the Swiss player six times.
Venus had been sidelined since losing the Key Biscayne final a month ago.
"I think it was a pretty good performance. I felt good on court," Venus said. "She probably likes clay with her type of game and I was able to execute pretty well out there on the court."
The sisters, former Rome champions, are preparing for the French Open, which begins on May 23.
In other results Tuesday, second-ranked Caroline Wozniacki dispatched Italian Maria Elena Camerin 6-1, 6-0 in less than one hour in the evening match, while former French Open champion Ana Ivanovic upset Victoria Azarenka, the ninth seed from Belarus, 6-4, 6-4. Azarenka appeared to be struggling with an injury to her leg, limping at times during the match.
Ivanovic's ranking has slipped from No. 1 to No. 58. The Serb has been trying to regain her form and recently hired Steffi Graf's old coach, Heinz Gunthardt.
The third-seeded Safina struggled for 2 hours, 40 minutes with her opponent and her own erratic game.
She recovered from 4-0 behind to level at 4-4 but went on to lose the next two games and the set to Dulgheru. After taking the second set in a tiebreaker, Safina had a disastrous third set, firing mistakes from both sides of the clay court.
Finding herself down 5-0, she twice smashed her racket in frustration. She sent a forehand wide to give the match to Dulgheru, who hadn't beaten anyone in the top 30 until now.
"In a second I was 5-0 after winning the second set," Safina said. "I think the only good thing about this match was that my back didn't create any problems."
A recent No. 1, Safina retired from the Australian Open in January with a lower back injury, and in her first tournament back _ last week's Porsche Grand Prix _ she lost in the quarterfinals.
Also on Tuesday, two-time former champion Jelena Jankovic defeated Bethanie Mattek-Sands of the United States 6-3, 3-6, 6-1, sixth-seeded Elena Dementieva of Russia beat Kazakh player Yaroslava Shvedova 6-4, 7-5 and Lucie Safarova of the Czech Republic brushed aside local hope Flavia Pennetta 6-1, 6-2. Eighth-seeded Agnieszka Radwanska beat Roberta Vinci of Italy 6-1, 6-0, and Shahar Peer of Israel came from behind to beat Polona Hercog of Slovenia 2-6, 7-5, 6-3.