Mitogen-stimulated human lymphocytes have an increased capacity to repair many forms of DNA damage caused by UV, ionizing radiation, and chemical carcinogens. Human lymphocytes rely on a particular DNA repair protein, O6-alkylguanine-DNA alkyltransferase (alkyltransferase) to repair efficiently O6-alkylguanine, an important mutagenic adduct formed by nitrosoureas and other N-nitroso compounds. The alkyltransferase is a "suicide" protein which becomes inactivated during the repair process. Thus, basal activity and the ability to synthesize new protein activity are important compounds of O6-alkylguanine repair. We compared basal and regenerated alkyltransferase activity in resting and mitogen-stimulated human lymphocytes. During stimulation with L-phytohemagglutinin, alkyltransferase activity increased by a mean of 70% over resting cells. Following exposure to N-nitroso-N-methylurea (MNU), alkyltransferase activity was consumed in a dose-dependent manner in both resting and L-phytohemagglutinin-stimulated cells by the repair of MNU-induced O6-methylguanine-DNA adducts. Recovery of alkyltransferase activity began within 1 day of MNU exposure in the L-phytohemagglutinin-stimulated lymphocytes but did not occur in resting cells. Enzyme induction was not observed. When the alkyltransferase was only partially inactivated by low dose MNU, resting lymphocytes still failed to recover alkyltransferase activity. The rate of recovery of alkyltransferase activity in proliferating cells was dependent on the basal level of activity, which varied about 3-fold among donors. These data indicate that mitogen-stimulated lymphocytes develop an increased capacity to repair nitrosourea-induced DNA damage and are able to regenerate activity following nitrosourea exposure. In contrast, resting lymphocytes do not rapidly synthesize new alkyltransferase molecules after nitrosourea exposure and appear susceptible to DNA damage caused by persistent O6-alkylguanine adducts. Thus, both basal alkyltransferase activity and the proliferative state of normal lymphocytes influence the response to nitrosourea exposure.