Abstract A radioisotopic, enzymatic cycling procedure was used to measure NAD, NADH, NADP and NADPH in cultured human lymphocytes at 0, 24 and 48 h after exposure to phytohemagglutinin (PHA). During the 0–24 h period after PHA addition NAD and NADH were increased in both control and test cultures leading to a decrease in the NAD: NADH ratio. During the 24–48 h period increases in NAD and NADH occurred in test cultures in parallel with increased incorporation of [ 3H]TdR. No change in the NAD: NADH ratio was seen. The results indicate that the levels of NAD and NADH may be affected by the culture conditions and that increases in these compounds occur in stimulated cells during a time period in which DNA turnover is elevated and cell volume is increased but before extensive cell division.