We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O 6 -(2-hydroxyethyl) 2'-deoxyguanosine (O 6 -HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O 6 -HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5mM HENU resulted in mutation frequencies of 7.2+/-2.2x10 - 5 , 45.2+/-2.9x10 - 5 and 120.3+/-24.4x10 - 5 , respectively. Comparison of the mutation frequencies demonstrates that 1 and 5mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C->A:T transitions (52%) and a significant number of A:T->T:A transversions (16%). We propose that the observed G:C->A:T transitions are produced by the DNA alkylation product O 6 -HOEtdG. These results suggest that the formation of O 6 -HOEtdG by BCNU treatment contributes to its observed mutagenic properties.