Ligation of interleukin 2 (IL2) is known to regulate both protein tyrosine and serine/threonine phosphorylation. A family of leukocyte transmembrane proteins whose cytoplasmic domain exhibits intrinsic protein tyrosine phosphatase activity is collectively called CD45 and is identified by a set of common cell surface epitopes. Although CD45 is known to be a phosphoprotein, it is not known how phosphorylation specifically regulates its function. We therefore identified a cell line, the IL4-dependent line CTLL-2.4, in which CD45 could be phosphorylated in response to addition of IL2. These cells are a variant of an IL2-dependent murine cell line which were selected for long-term growth on IL4 but which retain the ability to proliferate on exposure to IL2. Incubation of CTLL-2.4 in low serum concentrations followed by stimulation with IL2 caused a three- to fivefold increase in the phosphorylation of CD45 in a time- and concentration-dependent manner. CD45 in non-stimulated cells contained one major tryptic phosphopeptide, whereas, after exposure of the cells to IL2, two new phosphopeptides were present in CD45. The pattern of IL2-induced phosphorylation was different from that found following addition of phorbol 12-myristate 13-acetate (PMA) to the cells. Although IL2 induced rapid and potent tyrosine phosphorylation in CTLL-2.4 cells, all of the basal and cytokine-activated phosphorylation of CD45 occurred on serine residues. The IL2-stimulated phosphorylation caused no change in the amount of cell surface CD45 and no alteration of its catalytic activity using an artificial tyrosine phosphorylated substrate-RCM-lysozyme. We speculate that the increase in phosphorylation of CD45 may modify its association with potential substrates. The differences in the phosphorylation patterns induced by IL2 and PMA further suggest that more than one kinase can use CD45 as substrate and that IL2 activates a protein serine/threonine kinase different from protein kinase C.