By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a [Cys.sup.358]Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in tong free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in viva and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.