The behavior of phosphoribosylformylglycinamidine ( FGAM ) synthetase (EC 6.3.5.3) activity was elucidated in normal and proliferating tissues and in murine and human neoplasms. Enzymic activity was measured in the 100,000 X g crude supernatant fluid prepared from tissue homogenates. The assay was based on coupling FGAM produced to diazotizable aminoimidazole ribonucleotide. In the crude extracts of normal rat liver and hepatoma 3924A, the apparent KmS of FGAM synthetase for formylglycinamide ribonucleotide, adenosine triphosphate and L-glutamine were 0.06, 1.5, and 0.03 mM, respectively. The liver and hepatoma 3924A FGAM synthetases were saturated at formylglycinamide ribonucleotide, adenosine triphosphate, and L-glutamine concentrations of 0.1, 7.0, and 0.5 mM, respectively; both enzymes had a pH optimum of 7.4. In the liver of normal adult rats, the FGAM synthetase activity was 7.2 to 10.7 nmol/hr/mg protein. The synthetase specific activity in hepatomas of slow and medium growth rates increased 1.2- to 2.2-fold, and in rapidly growing hepatomas it was elevated 3.2- to 5-fold over the values of the respective control normal livers. There was a positive correlation between the increase in synthetase activity and hepatoma proliferation rate. In rat tissues of high cell renewal activity, thymus, spleen, and testis, synthetase specific activity was 7.0-, 3.9-, and 3.3-fold higher than that of normal liver. In the 24- and 48-hr regenerating liver, FGAM synthetase specific activity was increased by 1.2- and 1.5-fold, respectively. In 5-day-old differentiating liver, specific activity was 202% of the adult value; when data were expressed per average cell, the activity was 55% of that of the adult liver. The markedly increased activity in the rapidly proliferating hepatomas appears to be more characteristic of neoplastic growth than of normal liver proliferation. FGAM synthetase activity was also increased in human renal cell carcinoma and hepatocellular and colon carcinomas to 1.4-, 2.7-, and 3.8-fold of the activity of the respective homologous normal and host tissues. The synthetase activity in the rapidly proliferating murine Lewis lung carcinoma was 9.6-fold that of the normal lung. The increased activity of FGAM synthetase should confer selective advantages to the cancer cells and marks this glutamine-utilizing enzyme as a potentially important target in the design of chemotherapy.