A purine nucleoside phosphorylase enzyme preparation, which catalyzed the general reaction ribose (deoxyribose) [Formula: see text] nucleoside (deoxynucleoside)+orthophosphate, was isolated from muscles of the lingcod (Ophiodon elongatus). The reaction was found to be about 85% in favor of nucleoside synthesis with 10 μM./ml. concentrations of reactants in the hypoxanthine–inosine system. With similar concentrations of reactants in the guanine–guanosine system the equilibrium was about 75% in favor of nucleoside synthesis, and with considerably higher concentrations of reactants in this system this percentage decreased markedly. Purified preparations were stable for over a year at −20 °C., contained two major protein components (zone electrophoresis and ultracentrifuge data), were rather heat labile, and possessed a maximum activity corresponding to liberation of 32 μM. orthophosphate/mg. protein N/hour. Evidence is given in support of the view that a single enzyme is responsible for the above reaction and that it possesses strict specificity for α-D-ribofuranose 1-phosphate and deoxyribose 1-phosphate. Of a large number of substituted purines investigated, only the following were active: hypoxanthine, xanthine, 6-mercaptopurine, 6-methylpurine, 8-azaguanine, guanine, adenine, and 2, 6-diaminopurine. Methods are given for the preparation, in good yield, of analytically pure dicyclohexylammonium salts of ribose 1-phosphate and deoxyribose 1-phosphate.