cAMP inhibits the proliferation of both normal peripheral blood B and T lymphocytes as well as the proliferation of a human neoplastic B precursor cell line (Reh). To positively show that this is mediated via the the catalytic subunit, C alpha, of cAMP-dependent protein kinase, stably transfected Reh cell lines overexpressing C alpha were established. This was achieved by transfection with a construct confering hygromycin resistance together with a zinc-inducible expression of C alpha from the human metallothionine promoter. C alpha transfected clones were shown to confer a 2- to 2.5-fold zinc-dependent increase in C alpha messenger RNA, immunoreactive C, and phosphotransferase activity. The growth rate of clones transfected with C alpha was retarded, and a zinc-dependent inhibition of cell proliferation was demonstrated in the presence of a small trigger dose of forskolin. In contrast, overexpression of the regulatory subunit I alpha had no effect on cAMP-dependent inhibition of cell proliferation. Furthermore, expression of mutant regulatory subunit I alpha AB, which renders cAMP-dependent protein kinase unresponsive to cAMP, clearly protected against that inhibitory effect of cAMP. These data provides evidence that activation of the C subunit (C alpha) of cAMP-dependent protein kinase mediates the inhibitory action of cAMP on cell proliferation in Reh cells.