The effect of Ca 2ฯฉ -binding protein regucalcin on Ca 2ฯฉ -ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 ยตM) in the enzyme reaction mixture led to a significant increase in Ca 2ฯฉ -ATPase activity. Regucalcin significantly stimulated ATP-dependent 45 Ca 2ฯฉ uptake by the microsomes. Thapsigargin (10 ฯช6 M), a specific inhibitor of microsomal Ca 2ฯฉ pump enzyme (Ca 2ฯฉ -ATPase), clearly inhibited regucalcin (0.5 ยตM)-increased microsomal Ca 2ฯฉ -ATPase activity. Liver microsomal Ca 2ฯฉ -ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 ยตM) in increasing Ca 2ฯฉ -ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10 ฯช2 %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca 2ฯฉ ionophore (A23187; 10 ฯช7 M). The present study demonstrates that regucalcin can stimulate Ca 2ฯฉ pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca 2ฯฉ -ATPase.