A number of new period gene (per) mutants were generated by in vitro mutagenesis and germ line transformation. Missense mutations were made at amino acid 589, which is altered in the 19 h short-period (per(s)) mutant, and insertion mutations were generated with peptides commonly used for epitope tagging. Most of these new per mutants had short behavioral rhythms. Flies with heteroallelic combinations of these new mutant per genes were found to have "hybrid" periods, i.e., they had values that were usually in between those of the individual alleles. These findings suggest that short-period per mutants are not unusual gain-of-function mutants but rather more traditional loss-of-function mutants that are unable to influence the circadian pacemaker in a proper manner. The data also suggest that the per protein may engage in important intermolecular interactions.