The computer-animated stars from the 1980s claimed the number one post with a solid $25.4m estimated launch through Warner Bros.
Kevin Munroe directed the story of four mutant fighters who try to thwart an evil force. This is the fourth instalment in the franchise since the last episode opened in 1991. Chris Evans and Sarah Michelle Gellar are among the voice talent.
'We are thrilled with TMNT's performance this weekend and look forward to building a successful franchise with Warner Bros,' Harvey Weinstein, co-chairman of the film's international distributor TWC, said.
300 fell to second place after two weeks on top, adding $20.5m for a majestic $162.4m running total that should see the adventure cross $200m within weeks.
Paramount's assassination thriller Shooter starring Mark Wahlberg opened in third place on $14.5m, two places ahead of New Line's $10.2m launch of the family adventure The Last Mimzy, directed by New Line chief executive Bob Shaye.
Three more new releases broke into the top 10. Fox Atomic's horror sequel The Hills Have Eyes 2 opened in seventh place on $10m, Sony's drama Reign Over Me with Adam Sandler and Don Cheadle followed one place behind on $8m, while Lionsgate's drama Pride opened in ninth on $4m.
After four weekends the comedy Wild Hogs ranks fourth on $123.8m for Buena Vista, while Premonition has taken just over $32m for TriStar and ranks sixth after two.
Overall the top 12 titles grossed a combined $125.7m, representing a 28% rise against the same period last year.
Next weekend's wide releases are: Buena Vista's animated romp Meet The Robinsons; and Paramount's ice skating comedy Blades Of Glory with Will Ferrell and Jon Heder.
Estimated Top 10 North America Mar 23-25, 2007
Film (Distributor)/International distribution/Estimated weekend gross/Estimated total to date
1 (-) TMNT (Warner Bros) TWC $25.4m -
2 (1) 300 (Warner Bros) WBPI $20.5m $162.4m
3 (-) Shooter (Paramount) PPI $14.5m -
4 (2) Wild Hogs (Buena Vista) BVI $14.4m $123.8m
5 (-) The Last Mimzy (New Line) NLI $10.2m -
6 (3) Premonition (TriStar) Hyde Park Int'l $10.1m $32.2m
7 (-) The Hills Have Eyes 2 (Fox Atomic) Fox Intl $10m -
8 (-) Reign Over Me (Sony) SPRI $8m -
9 (-) Pride (Lionsgate) Lionsgate $4m -
10 (4) Dead Silence (Universal) UPI $3.5m $13.2m