The cytosolic 5'-nucleotidase specific for IMP, GMP, and their deoxyderivatives has been purified approximately 1000 times from calf thymus. The enzyme, in the presence of a suitable nucleoside, can act as a phosphotransferase, catalyzing the transfer of the phosphate moiety from a nucleoside monophosphate donor to a nucleoside acceptor, thus operating as an interconverting activity. This phosphorylating activity has drawn the attention of several research groups because the cytosolic 5'-nucleotidase represents the only cellular enzyme able to phosphorylate inosine and guanosine analogs, which are not substrates of known cellular nucleoside kinases. In this paper, we report the kinetic parameters of the bifunctional enzyme and its response to variations in adenylate energy charge. The results seem to indicate that in the presence of physiological concentrations of ATP and phosphate, the enzyme behaves mainly as a phosphotransferase, its activity being dependent only on the availability of a suitable nucleoside.