Protein phosphorylation in intact S49 mouse lymphoma cells was studied by using high-resolution two-dimensional gel electrophoresis of proteins labelled with [35S]methionine or [32P]Pi. In wild-type cells substrates for cyclic AMP-stimulatable phosphorylation exhibited high basal phosphorylation; in mutant cells deficient in activities of either cyclic AMP-dependent protein kinase or adenylate cyclase, basal phosphorylation of most of these substrates was negligible. Analysis of tryptic phosphopeptides from proteins labelled with [32P]Pi in wild-type cells suggested that identical sites were phosphorylated under conditions of both basal and hormonally elevated concentrations of cyclic AMP. These results argue that most basal phosphorylation is a consequence of partial activation of cyclic AMP-dependent protein kinase and that this activation is attributable to basal concentrations of cyclic AMP. For the intermediate filament protein vimentin, basal phosphorylation was largely at a site distinct from that stimulated by increased cyclic AMP, and basal phosphorylation was not markedly different in mutant and wild-type cells. Vimentin phosphorylated at both sites was not observed. Cyclic AMP treatment resulted in enhanced phosphorylation at the cyclic AMP-specific site and decreased phosphorylation at the cyclic AMP-independent site.