Superactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment. Two highly homologous isoforms of PRS (PRS1 and PRS2), each encoded by a distinct X chromosome-linked locus, have been identified, and PRS1 and 2 cDNAs have been cloned. The entire 954-base pair translated regions of PRS1 and 2 cDNAs derived from cultured lymphoblasts and fibroblasts from two patients in whom purine nucleotide feedback resistance of PRS is associated with enzyme superactivity and neurodevelopmental defects were examined by direct sequencing after polymerase chain reaction amplification of PRS transcripts. Nucleotide sequences of PRS2 cDNAs from the patients and normal individuals were identical. In contrast, PRS1 cDNAs from the patients differ from normal PRS1 cDNA, each by a single base substitution. PRS1 cDNA from patient N. B. showed an A to G transition at nucleotide 341, corresponding to an asparagine to serine change at amino acid residue 113 of mature PRS1. A G to C transversion at nucleotide 547, indicating an aspartic acid to histidine change at amino acid 182, was found for PRS1 cDNA from patient S. M. Point mutations at the sites identified in the PRS1 cDNAs of the two patients were confirmed by the results of RNase mapping analysis. Normal, N. B., and S. M. PRS1 cDNAs were introduced into Escherichia coli BL21 (DE3)/pLyS, and recombinant N. B. and S. M. PRS1s showed the purine nucleotide feedback resistance phenotypes characteristic of PRS from patients' cells.