Binding of nucleotides to bovine seminal RNAase was studied by differential spectrophotometry and equilibrium dialysis. Cytidine 3'-phosphate, the reaction product of the hydrolytic, rate-limiting step of the reaction, was found to be capable, in contrast to related nucleotides, of discriminating between the two structurally identical active sites of the enzyme. Negative co-operativity, with a 'half-of-sites' reactivity, was found at lower concentrations of ligand, whereas at higher concentrations positive co-operativity was detected. These findings exclude that the non-hyperbolic kinetics previously reported for the hydrolytic step of the reaction are due to hysteretic effect. A model of mixed-type co-operativity is proposed for interpreting the binding data.