Heparin has been shown to be involved in the regulation of cellular Ca2+ by binding to many proteins with high affinity. Here we examined the effects of heparin on the plasma membrane Ca2+-ATPase from porcine brain synaptosome. Our results showed that heparin dramatically inhibited the ATP hydrolysis and Ca2+ uptake in the presence and absence of calmodulin. Together with controlled proteolysis by trypsin, we concluded that the calmodulin-binding domain of the plasma membrane Ca2+-ATPase was less important for the heparin inhibition. Excess phosphatidylserine was able to eliminate the heparin inhibition. We observed that Ca2+ affinity kept no obvious changes, but the ATP affinity of plasma membrane Ca2+-ATPase was apparently decreased in the presence of heparin. Our results indicated that heparin had little effects on ATP or Ca2+ binding sites of the enzyme. Published in 2003.