In order to define the mechanism for the enhancement by thymidine (dThd) of the antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine (3'-CTNU) in mice, we have investigated the effect(s) of dThd on the uptake of nitrosourea by L1210 cells in culture, DNA alkylation, and repair of the alkyl lesion. Using a rapid centrifugation technique through silicone:paraffin oil, we observe a 1.3- and 1.5-fold increase in the uptake of radioactivity from 0.1 mM [chloroethyl-14C]BCNU in the presence of a 5- and 25-fold excess of dThd, respectively. Similarly, an enhancement of DNA alkylation was observed upon treatment of L1210 cells for up to 3 h with 0.1 mM [chloroethyl-14C]BCNU from 70 pmol 14C/mg DNA in control to 85, 95, and 120 pmol 14C/mg DNA with equimolar 5- and 25-fold excess dThd, respectively. No effect of dThd on the uptake of 0.1 mM [chloroethyl-14C]-3'-CTNU was observed, although a small increase in DNA alkylation at 3 h was evident. DNA repair, as measured by the amount of radioactivity remaining associated with the DNA after an initial 2-h treatment with labeled BCNU was largely unaffected by dThd. Although dThd appears to enhance the cellular uptake of BCNU and the alkylation of DNA by both BCNU and 3'-CTNU, dealkylative repair proceeds unhindered in the presence of dThd.