CD45 is a leukocyte-specific, transmembrane protein tyrosine phosphatase (PTPase) required for T cell responsiveness. How the activity of PTPases is regulated in vivo is unclear. Treatment of murine thymocytes and a variety of murine T cell lines with the calcium ionophore ionomycin decreased CD45 PTPase activity. Ionomycin treatment also led to a decreased phosphorylation of serine residues in CD45. These results indicate that increased intracellular calcium modulates CD45 PTPase activity, demonstrating regulation of CD45 PTPase activity in vivo, and also implicate serine dephosphorylation as a possible mechanism.