The effect of expression time (the time between the end of the treatment period and the first exposure to selective medium) on the frequency of benzo[a]pyrene (BP)-induced mutations at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase 2.4.2.8) locus was examined in the cell-mediated mutation assay in which V79 Chinese hamster cells are the targets and BP activation is mediated by Syrian hamster embryo cells. After the optimal expression time (4-5 days), the mutation frequency induced by exposure to 0.5 microgram BP/ml decreased logarithmically with increasing length of expression time until at 40 days there was a complete absence of mutant cells. The growth rates of three BP-induced 6-thioguanine-resistant clones were examined; each of these clones had a 30% longer lag time before reaching the period of logarithmic growth than the parent V79 cells. This suggests that the decay in mutation frequency with increasing expression time may be due to the slower reinitiation of growth by mutant than non-mutant cells after subcultivation into non-selective medium; this results in a loss of 25-40% of the mutant cells with each subculture. These findings show that optimal expression time in cell-mediated mutation assays may be of short duration and that this must be taken into account in the design and interpretation of such assays.