Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of 32P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem. 250, 4007-4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of 32P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42 000, 42 000, 80 000 and 90 000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t( 1/2 ) = 5-12s at 25°C. Between 40 and 70% of the 32P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function.