To determine if neuroblastoma acquires a sustained drug-resistant phenotype from patient exposure to therapy, we studied neuroblastoma cell lines established at different points of therapy: at diagnosis prior to therapy, at progressive disease after induction therapy and at relapse after intensive chemoradiotherapy and bone marrow transplantation (post-BMT). Melphalan, cisplatin, carboplatin, doxorubicin, and etoposide cytotoxicities were determined by DIMSCAN assay. Drug resistance progressively increased with therapy and 3/5 post-BMT lines showed high resistance to most drugs. IC 90s 37, 78, 719 and 256 times higher than clinically achievable drug levels were obtained in post-BMT cell lines for melphalan, cisplatin, doxorubicin and etoposide, respectively. Resistance correlated with the therapies patients received: considerable etoposide and doxorubicin resistance (> 1000-fold resistance) was seen in cell lines obtained from patients treated with these drugs. These cell lines indicate that neuroblastoma acquires resistance to cytotoxic drugs that is probably due to stable genetic alterations occurring during therapy.