In a prior report (Pike, L.M., Khym, J.X., Jones, M.H., Lee, W.H., and Volkin, E. (1980) J. Biol. Chem. 255, 3340-3347), it was observed that CTP synthesis and concomitant incorporation of CMP into RNA and dCMP into DNA were markedly reduced in cells cultured in the presence of cycloheximide and puromycin. Experiments described here with Novikoff hepatoma cells reveal that the purine biosynthetic pathway is similarly affected. When the cells are subjected to cycloheximide (30 or 60 microgram/ml) or puromycin (100 microgram/ml), there is a substantial reduction in the bioconversion of hypoxanthine, adenosine, and deoxyadenosine into guanylate compared to untreated cultures. Whereas synthesis (counts per min/nmol) of pool ATP was 70 to 100% of controls, that of pool GTP was 20 to 35% of controls. Incorporation of AMP into RNA was 40 to 60% of controls, but that of GMP was only 10 to 25% of controls. Incorporation of dAMP into DNA averaged 10% of controls, but that of dGMP was only 4% of controls. Synthesis of guanylates from formate by the de novo pathway was similarly reduced, but incorporation of guanosine, which enters via kinase action alone, was not disproportionately lowered. These results suggest that protein synthesis inhibitors cause a severely reduced availability of newly synthesized GTP and CTP as well as their deoxy counterparts, dGTP and dCTP, the proximal precursors for the synthesis of RNA and DNA. However, the nanomolar levels of all nucleoside triphosphates remain high, probably as a result of recycling of nucleic acid breakdown products. Thus, reduced synthesis of these compounds may restrict nucleic acid synthesis only if some sort of compartmentation leads to a limitation of these precursors at the site(s) of nucleic acid synthesis.