We have isolated a new human head and neck carcinoma cell line (C-10E) that is highly resistant to BLM (40-fold) when compared to the parental (A-253) cell line. Consonant with BLM resistance in the C-10E cell line, we found that this cell line accumulated 2- to 3-fold less BLM A2 than A-253 cells. Kinetic analyses of BLM A2 association revealed a decreased Vmax for C-10E cells with little change in Ka. Furthermore, the BLM-resistant cell line (C-10E) metabolized BLM A2 to a greater extent than its sensitive counterpart (A-253). Thus, compared to A-253 cells, the C-10E cells exhibited both decreased cellular association and increased metabolism of BLM. Synergistic cytotoxicity was seen when BLM was combined with either E-64 or leupeptin, cysteine proteinase inhibitors known to block BLM metabolism in vitro. E-64 inhibited the metabolism of BLM A2 in both C-10E and A-253 cells, and cellular accumulation of radiolabeled BLM A2 was increased by leupeptin or E-64 in only A-253 cells. These results suggest that both inhibition of drug metabolism and increased drug accumulation contribute to this synergism.