Disruptions of a Drosophila gene encoding a regulatory subunit of cAMP-dependent protein kinase homologous to mammalian RIbeta (dPKA-RI) were targeted to the first (noncoding) exon of dPKA-RI via site-selected P element mutagenesis. Flies homozygous for either of two mutant alleles showed specific defects in olfactory learning but not in subsequent memory decay. In contrast, olfactory acuity and shock reactivity, component behaviors required for normal odor avoidance learning, were normal in these mutants. Northern and Western blot analyses of mRNA and protein extracted from adult heads have revealed a complex lesion of the PKA-RI locus, including expression of a novel product and over- or underexpression of wild-type products in mutants. Western blot analysis revealed reductions in RI protein in mutants. PKA activity in the absence of exogenous cAMP also was significantly higher than normal in homogenates from mutant adult heads. These two mutant alleles failed to complement each other for each of these phenotypic defects, eliminating second-site mutations as a possible explanation. These results establish a role for an RI regulatory subunit of PKA in Pavlovian olfactory conditioning.