We found a new clock mutation,ritsu (rit), in an inbred strain separated from a natural population ofDrosophila melanogasterin Yamaguchi, Japan. This mutation lengthens the free-running period of the wild-type locomotor activity rhythm from ca. 24 hr to ca. 27 hr by lengthening both active and rest phases equally. To determine the loci involved, we established eight lines of chromosomal substituted flies between the inbred strain andCanton-S, and found that a major gene is located on the second chromosome. Furthermore, the result of recombination analysis suggested that the locus might be near or to the left ofcl(2-18.8).