We have explored the role of calmodulin in plasma membrane-related phenomena in lymphocyte activation by measurement of [125I]calmodulin binding to highly purified plasma membrane of human peripheral blood lymphocytes. Calcium-dependent calmodulin binding to lymphocyte membrane was found to reach equilibrium within 5 min of incubation at 37 degrees C and to be saturable and specific. A single class of high affinity-binding sites was identified, with a dissociation constant (Kd) of 1 to 3 X 10(-8) M and a total binding capacity (Bt) of 1 to 2 pmol/mg membrane protein. The free calcium concentration necessary for half-maximal binding was 100 to 300 nM. This was strikingly similar to the cytoplasmic-free calcium activity [Ca2+]i measured by the Quin-2 fluorescence technique, particularly after stimulation with phytomitogens. Calmodulin binding was inhibitable by trifluoperazine (TFP), W-7, and chloropramazine, all of which are calmodulin antagonists. The concentration of TFP that caused 50% inhibition of lymphocyte proliferative responses to phytomitogens was found to be identical to the concentration of TFP which causes 50% inhibition of calmodulin binding to lymphocyte plasma membrane. SDS-polyacrylamide gel electrophoresis followed by gel overlay and autoradiography with iodinated calmodulin revealed five calcium-dependent, TFP-inhibitable, calmodulin-binding polypeptides.