Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were $ 2.0 * 10^{-4}$ M for inosine, $2.0 *10^{-3}$ M for deoxyinosine, $ 2.0 * 10^{-5}$ M for guanosine and $2.0 10 ^{-5}$ M for deoxyguanosine. According to the ratio of relative $K_{cat}$Km, substrate specificity of each nucleoside was in the order of guanosine or deoxyguanosine, inosine and deoxyinosine. Cosubstrate, phosphate, revealed downward curvature in Lineweaver-Burk plot at high concentrations, indicating a negative cooperativity between subunits. The inhibition constants for purine analogs were measured to be $ 6 * 10^{-4}$ M for formycin B as the competitive inhibitor of inosine, $ 9 * 10^{-6}$ M for guanine as the competitive inhibitor of guanosine, $2 * 10^{-4}$ M for hypoxanthine as the non competitive inhibitor of guanosine and $4.5 * 10 ^{-4}$ M for 6-mercaptopurine as the non competitive inhibitor of guanosine. Alternative substrates, guanosine, deoxyguanosine and adenosine were found to act as competitive inhibitors with Ki values o $f^ 2.0 * 10 {-5}$ M, $2.6 * 10^{-5}$ M and $8.5 * 10 ^{-4}$ M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of $1.8 * 10^{-5}$ M and $ 3.0 * 10^{-5}$ M, respectively, when deoxyinesine was the variable substrate. The results of alternative substrate sstudies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine.e.