Of the mutationally defined rhythm genes in Drosophila melanogaster, period (per) has been studied the most. We have molecularly characterized three older per mutants-perT, perClk, and per04-along with a novel long-period one (perSLIH). Each mutant is the result of a single nucleotide change. perT, perClk, and perSLIH are accounted for by amino acid substitutions; per04 is altered at a splice site acceptor and causes aberrant splicing. perSLIH exhibits a long period of 27 hr in constant darkness and entrains to light/dark (L/D) cycles with a later-than-normal evening peak of locomotion. perSLIH males are more rhythmic than females. perSLIH's clock runs faster at higher temperatures and slower at lower ones, exhibiting a temperature-compensation defect opposite to that of perLong. The per-encoded protein (PER) in the perT mutant cycles in L/D with an earlier-than-normal peak; this peak in perSLIH is later than normal, and there was a slight difference in the PER timecourse of males vs. females. PER in per04 was undetectable. Two of these mutations, perSLIH and perClk, lie within regions of PER that have not been studied previously and may define important functional domains of this clock protein.