Spontaneous mutagenesis is thought to play a crucial role in spontaneous carcinogenesis. We recently described a new mathematical model for estimation of the spontaneous mutation rate (mutation/gene/generations) based on the assumption that mutations are fixed in the S-phase of the cell cycle. With this definition, the spontaneous mutation rate should be independent of the growth rate. In the present study, we tested this hypothesis, using cell line G12, a transgenic Chinese hamster V79 derivative, which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis. The growth rate was modulated by varying the serum concentration or the seeding density, or by addition of suramin, transforming growth factor beta, or dichlorobenzimidazole riboside to the medium. Significant increases in the spontaneous mutation rate occurred when cell proliferation was blocked by serum deprivation. Density-dependent inhibition of growth and inhibition of growth by suramin, transforming growth factor beta, or dichlorobenzimidazole riboside did not result in significant increases in spontaneous mutation rates. The level of oxidants in cells cultivated in the presence of low concentrations of serum was higher compared to control cells, suggesting that the increases in the spontaneous mutation rates under low serum conditions may be partly a result of oxidative stress due to a lack of serum antioxidants. This was shown to be the case, because spontaneous mutation rates were significantly reduced in serum-depleted cells when antioxidants were added to the medium. We suggest that during carcinogenesis, when tumors are in a prevascularized state, the spontaneous mutation rate may be elevated, and this process may contribute to the genetic instability of the tumor cells.