Glutamine (Gln) prevents apoptosis in intestinal epithelial cells but the mechanism(s) remain unknown. Gln-derived metabolites include ammonia, glutamate (Glu), glutathione (GSH), and nucleotides. We previously showed that Gln potently inhibited apoptosis in cytokine-treated human colonic HT-29 cells; this effect was specific to Gln, unaffected by Glu and unrelated to intracellular glutathione. The current research examines mechanism(s) for Gln-induced anti-apoptotic effects in HT-29 cells treated with tumor necrosis factor-alpha related apoptosis-inducing ligand (TRAIL). Proliferating cells were treated with Gln or selected Gln metabolites for 24 h. Cells were then treated with TRAIL and Gln or its downstream metabolites and apoptosis was assessed at 8 h after treatment. The purine and pyrimidine precursors inosine and orotate inhibited TRAIL-induced apoptosis. However, inhibition of purine synthesis with azaserine did not alter the potent anti-apoptotic effect of Gln. In contrast, the pyrimidine synthesis inhibitor, acivicin, completely prevented this response. Supplementation with the pyrimidine uracil or the pyrimidine precursor orotate rescued the acivicin-induced blockade of Gln anti-apoptotic action. Removal of bicarbonate, a substrate for pyrimidine synthesis, also inhibited the anti-apoptotic effects of Gln. Uracil and thymine alone also significantly decreased TRAIL-induced apoptosis. The anti-apoptotic effects of Gln were independent of DNA/RNA synthesis as measured by flow cytometry and BrdU incorporation. In conclusion, Gln prevents TRAIL-induced apoptosis in HT-29 cells through a mechanism involving the pyrimidine pathway. Our data also demonstrate the novel anti-apoptotic effects of pyrimidine bases and their precursor, orotate in these human intestinal cells.