Kinetic studies with substrates of the hydrolytic rate-limiting reaction step revealed that the non-hyperbolic kinetics of bovine seminal RNAse may not be ascribed to microheterogeneity of the enzyme or to hysteretic effects. The substrate saturation curves with intermediate plateau and the activating and inhibiting effects of the reaction product, respectively at low and high concentrations, are explained in terms of mixed co-operativity, with binding at subsites that is a prerequisite for full activity of the enzyme. A model is proposed that is supported also by the results of binding studies.