In situ hybridization was used to localize cAMP-dependent protein kinase (PKA) mRNAs in the adult mouse CNS. The PKA holoenzyme contains two catalytic (C) subunits and a regulatory (R) subunit dimer. Our studies demonstrate expression of two isoforms of C (C alpha and C beta) and four isoforms of R (RI alpha, RI beta, RII alpha, and RII beta) in the CNS. mRNAs for C alpha, RI alpha, and RI beta preferentially localize in the neocortex, caudate-putamen, hypothalamus, thalamus, and hippocampus. Hybridization with C beta and RII beta probes is clearly distinguished from the C alpha-like pattern by a reduced level of hybridization in the thalamus and by a relative increase in expression in the dentate gyrus compared with cell layers CA1-3 in the hippocampus. RII alpha transcripts are very specifically localized in the medial habenula. The differential expression of PKA subunit genes suggests that functional differences in cAMP responses within neural tissues may be mediated by the biochemical properties of specific PKA isoforms.