Primary cultures of rat urothelial cells were exposed to hydroxyurea, [3H]thymidine, and 4-nitroquinoline 1-oxide (NQO) or N-hydroxy-4-aminoquinoline 1-oxide (HAQO) in a serum-free media for 2 h; unscheduled DNA synthesis (UDS) was measured by autoradiography. Both NQO and HAQO produced unscheduled DNA synthesis. Dicumarol, an inhibitor of NQO nitroreductase, inhibited the activity of NQO and, to a lesser extent, HAQO. Pyrophosphate, an inhibitor of seryl-AMP synthetase, inhibited the activity of both compounds. Neither dicumarol nor pyrophosphate, under similar experimental conditions, inhibited the activity of N-hydroxy-N-2-acetylaminofluorene (N-OH-AAF). These results support the idea that nitro-reductase and seryl-AMP synthetase may be involved in the activation of NQO.