The 27-year-old American was overtaken in the rankings two weeks ago by Dinara Safina, making the Russian and older sibling Marat Safin the only brother-sister duo to have been No. 1 in professional tennis.
"We all know who the real No. 1 is,'' Williams said Monday at the Italian Open. "Quite frankly, I'm the best in the world.''
Safina has never won a Grand Slam title, losing last year's French Open final and being beaten in the Australian Open final this year by Williams.
"I'm excited for her,'' Williams said with a smile.
Safina acknowledged she is still coming to terms with her new position but insists it is well deserved.
"It's still tough to realize this,'' Safina said. "It's not luck. Nobody gave it to me - I proved it. I played two Grand Slam finals, and last year I won four titles ... so I think I belong to this, because this doesn't come out of sky.''
The 23-year-old Safina said Williams is entitled to claim to be the real No. 1 because she has won 10 Grand Slam titles.
"But she's also older than me, so she has more experience than me,'' Safina said. "So let's see when I'm her age how many titles I'm going to have, and then we can say.''
Williams, the 2002 Rome champion, opens against 2005 runner-up Patty Schnyder of Switzerland on Tuesday in her first clay-court event of the season.
When asked which rival poses the biggest threat, Williams said: "Probably myself. I always beat myself.''
Her sister Venus Williams, seeded fourth and on the opposite side of the draw to Serena, is also in Rome and scheduled to play Monday against Lucie Safarova of the Czech Republic.
Safina, the 2006 runner-up, two-time defending champion Jelena Jankovic of Serbia and Ana Ivanovic of Serbia are among the top eight seeded players who have first-round byes.
Also on NBCSports.com
Ventre: Lakers need killer instinct against Rockets
Celizic: Tipping pitches would be worst A-Rod deed
NHL: Highlights from the Capitals' Game 2 win
NFL: Who's happy, who's worried after draft?
Tennis: Can anyone beat Nadal on clay?
