The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca(2+)-requiring proteinase required 5-10 microM Ca2+ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25-2.0 microM) in the absence or presence of Ca2+ (5.0 microM) added. The effect of regucalcin, however, was the greater in the absence of Ca2+ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca2+ (5.0 microM). In the absence of Ca2+, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 micrograms/ml), and heavy metals (25 microM cadmium or 25 microM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca(2+)-independent neutral cysteinyl-proteinase.