The addition of anti-immunoglobulin (anti-Ig) to purified rabbit B-cells or concanavalin A (Con A) to purified rabbit T-cells within minutes resulted in the phosphorylation of a number of cytosolic proteins. Two-dimensional (2-D) electrophoresis and autoradiography of 32P-labeled cell sonicates was used to identify proteins whose phosphorylation was enhanced by these mitogens. Two proteins, pp58 and pp90, were phosphorylated 1.5 min after addition of anti-Ig to B-cells. Four other proteins, pp60, pp65, pp67 and pp95, were phosphorylated at later times. Three of these proteins were also phosphorylated after addition of Con A to purified T-cells. These phosphoproteins do not correspond to any previously described cytoplasmic proteins. Although all of these phosphoproteins were present in the cytosolic fraction, pp58 may be associated with the cytoskeleton. Protein pp58 is also distinguished from the rest by its absence from 2-D gels run under non-reducing conditions. Treatment of the B-cells with F(ab')2 fragments of anti-Ig stimulated phosphorylation but Fab' fragments did not--indicating that receptor cross-linking is required to induce phosphorylation. Both pp58 and pp90 contained phosphoserine, but neither phosphothreonine nor phosphotyrosine. Quantitatively the 32P-labeling of pp58 was 2.7-fold over background at 10 min after anti-Ig addition. The identification of these phosphoproteins, which may play a role in activational cascades or in cytoskeletal rearrangements, hopefully will help to clarify the interrelationships between cyclic nucleotide dependent and independent kinases in lymphocyte activation.