We investigated the cytotoxic and cytogenetic effects of 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea on five cell lines established from human glioma biopsy specimens. Compared to the sensitive cell line SF-126, SF-188 cells are 3- to 6.5-fold more resistant to the cytotoxic effects and 8- to 14-fold more resistant to the induction of sister chromatid exchanges. Cytotoxic effects and induction of sister chromatid exchanges are intermediate for SF-210 and SF-295 cell lines compared with SF-126 and SF-188. There is a good correlation between susceptibility to the cytotoxic effects and formation of DNA interstrand cross-links for cells treated with 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea . We quantitated the extent of repair of O6-methylguanine after treatment of these cell lines with [3H]methylnitrosourea. SF-126 cells showed no detectable repair of O6-methylguanine, SF-210 and SF-295 had intermediate levels of repair, and SF-188 had very high levels of repair. We conclude that the cellular capacity to repair O6-chloroethylguanine adducts in DNA, which is reflected in the methyl repair process, is an important factor in determining cytotoxic response, and that increased repair of O6-chloroethylguanine decreases cytotoxicity and causes fewer sister chromatid exchanges and DNA interstrand cross-links to form in cells treated with chloroethylnitrosoureas. We studied the effects of cis-diamminedichloroplatinum(II) and nitrogen mustard in these cell lines. cis-Diamminedichloroplatinum(II) was equally cytotoxic and induced the same number of sister chromatid exchanges and DNA interstrand cross-links in all five cell lines. In contrast to the results obtained by treatment with chloroethylnitrosoureas, SF-126 cells treated with nitrogen mustard are 7.6-fold more resistant to the cytotoxic effects, 2-fold more resistant to the induction of sister chromatid exchanges, and 3-fold more resistant to the induction of DNA interstrand cross-links than are SF-188 cells. The results of this investigation with five human glial-derived cell lines clearly indicate that the molecular mechanisms of cellular resistance to alkylating chemotherapeutic agents are highly specific. Cellular resistance to chloroethylnitrosoureas does not result in cross-resistance to nitrogen mustard or cis-diamminedichloroplatinum(II).