Major histocompatibility complex class H molecules and the B cell antigen receptor (BCR) transduce similar signals when cross-linked by ligand. Therefore, studies were conducted to determine whether the protein tyrosine phosphatase CD45 regulates signaling via these transmembrane receptors in an analogous manner. Cross-linking of either class II molecules or the BCR on CD45-positive K46-17 μmλ B lymphoma cells was observed to induce activation of the Src family protein-tyrosine kinase Lyn, tyrosine phosphorylation of Syk and phospholipase Cγ, and the production of inositol 1,4,5-trisphosphate leading to intracellular mobilization as well as extracellular influx of Ca2+. In the absence of CD45, cross-linking of either class II molecules or the BCR failed to induce activation of Lyn. Syk was inducibly phosphorylated on tyrosine in a normal manner, whereas phospholipase Cγ exhibited a high basal level of tyrosine phosphorylation that was not significantly increased upon stimulation. Nevertheless, phospholipase Cγ appeared to be functional because CD45-negative cells produced elevated levels of inositol 1,4,5- trisphosphate following stimulation through class II or the BCR. Regardless of this, CD45-negative cells exhibited Ca2+ mobilization responses that were greatly diminished and transient in nature. Whereas little or no mobilization of Ca2+ was observed in response to class II cross-linking, CD45-deficient cells mobilized Ca2+ from intracellular stores but not the extracellular environment in response to BCR cross-linking. These results demonstrate that CD45 regulates both Src family kinase activation and Ca2+ mobilization associated with class II- and BCR-mediated signal transduction.