We previously reported that triciribine (tricyclic nucleoside, TCN, NSC-154020), after phosphorylation in cultured CCRF-CEM human leukemic lymphoblasts inhibited de novo purine nucleotide synthesis, GTP more than ATP [Moore et al. Biochem. Pharmac. 38, 4037 (1989)]. To determine the enzymes inhibited, triciribine phosphate (TCN-P, NSC-280594) was tested in dialyzed extracts of the cells. A new assay for glycinamide ribotide (GAR) synthesis was based on incorporation of [14C]glycine into GAR as a ribose-containing compound retained on boronyl gel columns. Glutamine, phosphoribosyl pyrophosphate (PRPP), ATP and glycine were required for the two-step sequence of glutamine:amidophosphoribosyltransferase (EC 2.4.2.14) and phosphoribosylamine-glycine ligase (EC 6.3.4.13). When PRPP was near the normal intracellular concentration (0.1 mM), 1.2 mM TCN-P inhibited GAR synthesis by 71-95%. To permit separate assay of the ligase step, 6-diazo-5-oxo-L-norleucine was used to inhibit amidophosphoribosyltransferase and phosphoribosylamine (PRA) was supplied in situ by chemical reaction of ribose-5-phosphate and ammonia (as ammonium acetate). The ligase was not inhibited by TCN-P. Thus, TCN-P inhibits amidophosphoribosyltransferase; it acts as an analog of the purine nucleotides which regulate this first committed step of de novo purine biosynthesis by an allosteric feedback mechanism. The measured intracellular concentration (0.1 mM) of PRPP was not changed in cells treated with TCN. IMP dehydrogenase (EC 1.1.1.205), the first de novo step committed to guanosine nucleotide synthesis, was also tested. It was inhibited by TCN-P, competitively with IMP, 66% at 1.2 mM TCN-P and 8 microM IMP. The degree of inhibition of these two enzymes was sufficient to account for the effects on purine nucleotide biosynthesis observed in intact cells treated with TCN.