Abstract The role of regucalcin in the regulation of protein kinase activity in rat brain neuronal cells obtained from primary culture was investigated. Protein kinase activity was assayed using the 5500 g supernatant fraction of the cell homogenate. Protein kinase activity was significantly raised by the addition of calmodulin (5 μg/ml) or dioctanoylglycerol (5 μg/ml) in the presence of CaCl 2 (10 −4 M), indicating that Ca 2+/calmodulin-dependent protein kinase and protein kinase C is present in the neuronal cells. The addition of regucalcin (10 −9–10 −7 M) in the enzyme reaction mixture caused a significant decrease in protein kinase activity in the absence of calmodulin or dioctanoylglycerol without Ca 2+ addition. Moreover, regucalcin completely prevented the activation of protein kinase by the addition of calmodulin or dioctonoylglyceral in the presence of CaCl 2 (10 −4 M). The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) caused a significant elevation of protein kinase activity without CaCl 2 addition. Such an effect was significantly inhibited by the addition of trifluoperazine (2×10 −5 M), an antagonist of calmodulin, or staurosporine (10 −6 M), an inhibitor of protein kinase C. The present study demonstrates that endogenous regucalcin in rat brain neuronal cells has an inhibitory effect on Ca 2+-dependent protein kinase activity.