Purine nucleoside phosphorylase (PNP) was purified to homogeneity. The molecular weight of the enzyme was 170,000. The enzyme consisted of six subunits, each with a molecular weight of 27,000. Serratia PNP had ten times the affinity for adenosine and deoxyadenosine than for inosine and deoxyinosine in a pattern characteristic of bacterial PNP. 1-Methylinosine and 1-methylguanosine, which have no affinity for mammalian PNP, bound Serratia PNP. In terms of Kcat/K(m), the substrate specificities were in the descending order of guanosine, inosine, and adenosine. When inosine or deoxyinosine was used as a variable substrate, a biphasic reciprocal plot with upward curvature was observed. The values of the Hill coefficient were 1.2 and 1.1 for inosine and deoxyinosine, respectively. Positive cooperativity seemed to be involved in the binding of inosine and deoxyinosine to the enzyme.