Values of inhibition constants, Ki, for a family of structurally related, competitive inhibitors of calf spleen purine nucleoside phosphorylase (PNP) have been determined employing both inosine as substrate and a manual assay and 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) as substrate and a robot-based enzyme kinetics facility. Several of the values determined robotically were confirmed employing the same substrate and a manual assay. Surprisingly, for many of the inhibitors examined, values of Ki determined with MESG as substrate are smaller than those obtained employing inosine as substrate by a factor that varies from less than 2 to 10. Values of concentrations required for 50% inhibition of PNP, IC50, have also been determined for the same family of inhibitors employing inosine as substrate. Values of IC50ino and those for Kiino and Kimesg for subsets of the inhibitors have been employed as training sets to create quantitative structure-activity relationships (QSAR) which have substantial power to predict values of IC50 and Ki for inhibitors outside the training set. These QSAR models should be useful in guiding future medicinal chemistry efforts designed to discover inhibitors of PNP having increased potency.