As a consequence of its central role in the regulation of calcium metabolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was assessed for cAMP-dependent and tyrosine phosphorylation. Addition of forskolin or prostaglandin E1, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [32P]PO4 resulted in the direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes resulted in a 1.4-fold stimulation of activity. Thus, the previously reported inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting in a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting platelets exhibited little tyrosine phosphorylation. Phosphorylation of platelet plasma membranes by pp60(src) resulted in 75% inhibition of PMCA activity within 15 min. Similarly, membranes isolated from thrombin-treated platelets exhibited 40% lower PMCA activity than those from resting platelets. Phosphorylation of erythrocyte ghosts and purified PMCA by pp60(src) also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation. Sequencing of a peptide obtained after 32P labeling of purified erythrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphorylated by pp60(src). These results indicate that tyrosine phosphorylation of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.