A deficiency of purine nucleoside phosphorylase activity is associated with marked depletion of T-lymphocytes which is felt to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. Human T-lymphoblasts incubated in the presence of 2'-deoxyguanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphate whereas B-lymphoblasts and mature T4+-cell lines accumulate GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a complete block at the G1-S interface of the cell cycle. In contrast 3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine incorporation into DNA or RNA but do cause a pronounced slowing in the progression of cells through S phase. B-lymphoblasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-triphosphate from 2'-deoxyguanosine and progress normally through the cell cycle, demonstrating a requirement for guanine salvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide pools but not with inhibition of protein synthesis or alterations in basal levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic guanosine monophosphate. We conclude that the accumulation of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and its analogues.