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It is well documented that the b-gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N-terminal end encoded by alternative use of several exons located 5¢ to exon 2 in the human, bovine and mouse Cb gene. In the present study, we demonstrate the expression of six novel human Cb mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbD4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbD4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbD4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP-responsive element binding protein. Furthermore, co-expression of CbD4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CbD4 variants associate with both RI and RII in a cAMP-independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CbD4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.