PRPP synthetase catalyzes the synthesis of PRPP, a regulatory substrate in the pathway of purine nucleotide synthesis de novo. We have developed a specific assay for quantitative determination of PRPP synthetase immunologically cross-reactive material in human erythrocyte and fibroblast extracts. The sensitivity of the radioimmunoassay (0.3% and 0.08% of normal mean cross-reactive material in erythrocytes and fibroblasts, respectively) was equivalent to that of the enzymatic activity assay, but enzyme protein initially present in relatively inactive monomeric and smaller aggregated forms was radioimmunochemically measurable. The radioimmunoassay was utilized in conjunction with the enzymatic assay to study normal PRPP synthetase and PRPP synthetases from five affected male patients (in four families) in whom inherited enzyme superactivity was associated with increased rates of PRPP and purine nucleotide synthesis and gout with excessive uric acid excretion. Despite increased enzymatic activities in patients' cell extracts, values for cross-reactive material were within the ranges measured in the respective normal cell extracts. Thus, calculated absolute specific activities (nmol/hr/mg cross-reactive material) of patients' PRPP synthetases were substantially greater than those of normal PRPP synthetase. Moreover, absolute specific activities in hemolysates from both patients and normal individuals were in close agreement with the enzyme-specific activities measured in preparations of erythrocyte PRPP synthetase purified to homogeneity from the corresponding patient or normal source. These findings provided evidence for the accuracy and specificity of the radioimmunoassay and supported previous evidence for increased maximal reaction velocity as the basis of superactivity of the patients' enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)