Oligonucleotides derived from the previously published rat testicular protein kinase inhibitor (PKI) sequence (Van Patten, S. M., Ng, D. C., Th'ng, J. P. H., Angelos, K. L., Smith, A. J., and Walsh, D. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5383-5387) were used to isolate a DNA fragment coding for a mouse homologue of the rat testicular PKI. This DNA fragment was then used to screen a mouse brain cDNA library, and two cDNA clones related to the testicular PKI (PKI beta) were isolated. Sequencing and comparison showed that the two cDNAs differ only in the presence of a 105-base pair insert, which results in an amino-terminal extension of the predicted PKI beta 2 protein by 20 residues relative to the PKI beta 1 protein. By using the appropriate primers, PCR was used to amplify specific regions of both of these clones from mouse brain cDNA. When both clones were expressed in vitro, the mRNA for PKI beta 2 produced a protein product that was larger and much more effectively translated, suggesting a functional role for the inserted sequence. Both isoforms were transiently expressed in COS-1 cells to evaluate their ability to inhibit the catalytic subunit of PKA in vivo. Extracts from cells expressing the PKI beta 2 isoform showed greater inhibition of catalytic subunit kinase activity than extracts expressing the PKI beta 1 isoform. Northern blot analysis of poly(A)+ RNA from various mouse tissues showed the presence of transcripts of 1.8 kilobases related to these cDNAs. Analysis of brain RNA from several species indicated that expression of these PKI mRNAs is evolutionarily conserved. Together with previous studies, these results indicate the presence of at least three PKI proteins in mouse. The skeletal muscle isoform has been designated PKI alpha, while the testicular isoform is PKI beta. We propose to designate the two testicular isoforms described here PKI beta 1 and PKI beta 2.