DNA repair of O6-alkylguanine by O6-alkylguanine-DNA alkyltransferase (O6-AT) may be crucial in modulating the extent of cytogenetic damage induced by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an important anticancer chemotherapeutic agent. To test this idea we treated normal human lymphocytes for 1 h with methylnitrosourea (MNU), which inactivates O6-AT, and then treated them for 1 h with BCNU. The result was a synergistic increase in the number of sister chromatid exchanges (SCEs) and chromosomal aberrations induced. BCNU-induced SCEs were potentiated 1.4- to 2.5-fold in MNU-pretreated cultures. Pretreatment of lymphocytes with MNU resulted in a 4- to 28-fold increase in the number of BCNU-induced chromatid aberrations. The enhancement of both SCEs and aberrations induced was MNU-dose dependent. Treatment of lymphocytes with MNU alone did not affect cell cycle progression, suggesting that MNU does not influence the extent of BCNU-induced cytogenetic damage by selecting highly damaged cells through alterations in lymphocyte proliferation kinetics. The potentiation of SCE and aberration induction by pretreatment with MNU is postulated to be due to inhibition of O6-AT. This inhibition would permit the formation and persistence of BCNU-induced O6-(2-chloroethyl)guanine monoadducts, which can undergo subsequent reactions to form DNA cross-links. In humans variations in O6-AT resulting from acute alkylation exposure or genetic deficiency may be important in modulating the genotoxic effects of BCNU.