Purine nucleotide and nucleic acid synthesis were studied in cultures of human first and third trimester trophoblastic cells. De novo synthesis was measured as incorporation of 14C-formate into purine nucleotides. Reutilization of purine bases was evaluated by the incorporation of 14C-adenine and -hypoxanthine. Utilization of 14C-adenine was also studied. The incorporation of formate was significantly (P less than 0.01) less active in the third trimester cells. Adenine incorporation was an order of magnitude higher than that of formate in both first and third trimester cells, and significantly (P less than 0.001) higher in the first than third trimester cells. No change in the reutilization of hypoxanthine was observed as a function of gestational age, and the rate was not increased by high extracellular inorganic phosphate. Both phosphorylation and deamination of adenosine increased as a function of concentration up to at least 60 microM, and the high concentration was more efficiently utilized in the first trimester cells. The major pathways of purine nucleotide synthesis function in the human trophoblast throughout gestation, but the contribution of reutilization seems larger than that of de novo synthesis. First trimester trophoblast appears more active in nucleotide and nucleic acid synthesis. Hypoxanthine utilization appears not be enhanced by increased extracellular phosphate. Hypoxanthine may be the major precursor utilized in trophoblastic purine nucleotide synthesis.