Cells must increase synthesis of purine nucleotides/deoxynucleotides before or during S-phase. We found that rates of purine synthesis via the de novo and salvage pathways increased 5.0- and 3.3-fold respectively, as cells progressed from mid-G1-phase to early S-phase. The increased purine synthesis could be attributed to a 3.2-fold increase in intracellular PRPP (5-phosphoribosyl-α-1-pyrophosphate), a rate-limiting substrate for de novo and salvage purine synthesis. PRPP can be produced by the oxidative and non-oxidative pentose phosphate pathways, and we found a 3.1-fold increase in flow through the non-oxidative pathway, with no change in oxidative pathway activity. Non-oxidative pentose phosphate pathway enzymes showed no change in activity, but PRPP synthetase is regulated by phosphate, and we found that phosphate uptake and total intracellular phosphate concentration increased significantly between mid-G1-phase and early S-phase. Over the same time period, PRPP synthetase activity increased 2.5-fold when assayed in the absence of added phosphate, making enzyme activity dependent on cellular phosphate at the time of extraction. We conclude that purine synthesis increases as cells progress from G1- to S-phase, and that the increase is from heightened PRPP synthetase activity due to increased intracellular phosphate.