In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt(S)) and long-period (dbt(L)) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt(S) and dbt(L) mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT(S), and DBT(L) proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt(S) and dbt(L) mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKIdelta expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt(S) and dbt(L) mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.