A panel of murine monoclonal antibodies (MAbs) was produced by fusing NS-0 myeloma cells with spleen cells of a BALB/c X DBA/2 F1 mouse hyperimmunized against a highly immunogenic subline of the L1210 leukemia obtained by in vivo treatment of the L1210 parental line with the antitumor drug 5-(3,3'-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). Among the 52 MAbs produced 16 (anti-D) were specifically cytotoxic in a complement-dependent cytotoxicity assay for the drug-altered subline and the others (anti-L) also cross-reacted with the L1210 parental leukemia. Six anti-D and three anti-L MAbs were selected for detailed studies of tissue specificity. In quantitative absorption experiments the antigens defined by these antibodies could not be detected on cells from normal mouse tissues (lung, liver, kidney, heart, spleen, and thymus). The reactivities of both anti-D and anti-L MAbs against a panel of L1210/DTIC sublines obtained at different times were assayed. The results showed that the antigenic specificities defined by anti-L MAbs were expressed on almost every L1210/DTIC subline while the anti-D MAbs detected antigenic structures specific for the L1210/DTIC used for the immunization. None of the MAbs tested cross-reacted with the L5178Y lymphoma or with its DTIC-altered sublines. The failure of anti-D MAbs to cross-react with cells from other L1210/DTIC sublines supports the hypothesis that the immunological alterations induced by the DTIC treatment are the consequence of mutagenic activity of the drug. On the other hand the presence of anti-L antigens on the cells of every L1210 subline indicates that the DTIC alteration is not accompanied by a loss of the tumor-associated antigen from the L1210 leukemia.