Mouse genes for two isozymes of the catalytic subunit of cAMP-dependent protein kinase (C alpha, C beta) were isolated and characterized. The C alpha gene is divided into 10 exons contained on three overlapping clones. Genomic clones containing the 5' end of the C beta gene were also isolated, and sequence analysis demonstrated that the positions of the three introns examined are identical in the C alpha and C beta genes. S1 mapping and primer extension experiments indicate that transcription initiated from multiple sites in both genes and that neither contain recognizable TATA or CAAT homologies. The 5' flanking regions of both genes are remarkably high in GC content (80% for C alpha and 72% for C beta) and are similar to the "HTF" (HpaII tiny fragment fraction) island sequences which have recently been found to flank several other mammalian transcriptional units.