A number of newperiodgene (per) mutants were generated byin vitromutagenesis and germ line transformation. Missense mutations were made at amino acid 589, which is altered in the 19 h short-period (peras) mutant, and insertion mutations were generated with peptides commonly used for epitope tagging. Most of these newpermutants had short behavioral rhythms. Flies with heteroallelic combinations of these new mutantpergenes 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-periodpermutants 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 theperprotein may engage in important intermolecular interactions.