We have previously identified and characterized regulatory (R) subunits of cyclic AMP-dependent protein kinase, particularly the RII subunits in rat tissues (Jahnsen, T., Lohmann, S. M., Walter, U., Hedin, L., and Richards, J. S. (1985) J. Biol. Chem. 260, 15980-15987; Jahnsen, T., Hedin, L., Lohmann, S. M., Walter, U., and Richards, J. S. (1986) J. Biol. Chem. 261, 6637-6639; Jahnsen, T., Hedin, L., Kidd, V. J., Beattie, W. G., Lohmann, S. M., Walter, U., Durica, J., Schulz, T. Z., Schiltz, E., Browner, M., Lawrence, C. B., Goldman, D., Ratoosh, S. L., and Richards, J. S. (1986) J. Biol. Chem. 261, 12352-12361). These studies showed that rat RII alpha and RII beta had apparent molecular masses of 54 and 52 kDa, respectively. The aim of the present study was to purify and characterize cAMP-dependent protein kinase R subunits in human testis and to examine which of the subunits (mRNAs and proteins) are present in this tissue. Our results show that human testis contains mRNAs for five out of the seven known subunits of cAMP-dependent protein kinase. We observed strong expression of mRNAs for RI alpha (1.5 and 3.2 kilobases (kb)), RII alpha (2.2, 2.4, and 7.0 kb), and RII beta (3.3 kb). We also demonstrated mRNAs for two of the three catalytic subunits, C alpha (2.7 kb) and C gamma (1.7 kb). Purification of R subunits by DEAE-cellulose and cAMP affinity chromatography revealed three distinct forms with apparent molecular masses of 49, 51, and 53 kDa, respectively. Characterization of these R subunits by their 8-azido-cAMP photoaffinity labeling and immunoreactivity, as well as by a phosphorylation-dependent mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated subunit sizes of RII beta (53 kDa) greater than RII alpha dephosphoform (51 kDa) greater than RI alpha (49 kDa). This conclusion was verified by the analysis of RII subunits produced by in vitro transcription/translation of full-length cDNAs for both human RII alpha and RII beta in wheat germ lysates. The in vitro translated products were the same size as the purified human testis subunits, and only the smallest RII subunit (RII alpha) revealed a distinct mobility shift on SDS-PAGE after phosphorylation/dephosphorylation. This study supports the conclusion that the mobilities of human RII subunits (RII alpha, RII beta) on SDS-PAGE are reversed in contrast with those of other species such as rat and bovine.(ABSTRACT TRUNCATED AT 400 WORDS)