A molecular cDNA clone (KIN27) was isolated that encodes a novel variant of the catalytic subunit C alpha of the human cyclic AMP-dependent protein kinase (PKA). Analysis of PKA genomic sequence data revealed that the KIN27 variant transcripts likely result from alternative splice-site selection of the C alpha gene transcripts. The KIN27 cDNA has therefore been designated C alpha 2. RNA amplification by polymerase chain reaction revealed that KIN27 C alpha 2 splice-site variant RNA and PKA C alpha RNA were both expressed in the amnion U and HeLa human cell lines. C alpha RNA was about 3- to 5-fold more abundant than C alpha 2 RNA. Interferon treatment decreased the steady-state amount of the C alpha 2 RNA relative to C alpha RNA. These results suggest that alternative splicing may contribute to the structural heterogeneity of C subunits expressed in human cells and that interferon may affect this process.