Insulin-degrading neutral proteinase with molecular weight of 70 kDa was partly purified from the rat liver and erythrocyte plasma membranes. Incubation of membranes with [gamma-32P]ATP resulted in the enzyme phosphorylation. Intensity of this process greatly increased in the presence of insulin (100 microU/ml), and correlated with the elevation of the insulin-degrading activity in proteinase. Ca2+, Mn2+, dithiothreitol, cysteine were shown to have a stimulatory effect on insulin degradation; p-chloromercuribenzoate significantly repressed this process. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor did not affect the activity of the proteinase. It was concluded that the investigated enzyme was a calpain and may participate in the mechanism of insulin action.