Background: The clinical effect of chemotherapy is dependent on drug pharmacokinetics, cellular response and regenerative potential of residual cells. Objective: Analysis of in vitro drug resistance in childhood acute lymphoblastic leukaemia (ALL) on diagnosis and at relapse, with respect to cell cycle study and gene rearrangements. Material and methods: A total of 116 patients entered the study, including 106 de novo and 23 relapsed children; in 13 patients paired analysis was performed. In vitro drug resistance profile for 21 compounds was done by the MTT assay, including combined drug resistance profile for prednisolone, vincristine and L-asparaginase (PVA score). DNA index and cell cycle phases were analyzed by flow cytometry. Gene rearrangement study was done by FISH and PCR. Results: PVA score showed higher in vitro drug resistance of lymphoblasts at relapse and in T-ALL. Relapsed lymphoblasts were more in vitro resistant to prednisolone, dexamethasone and thioguanine. Paired analysis showed good sensitivity at relapse for cytarabine and cladribine. T-ALL lymphoblasts were more in vitro resistant than common-ALL to vincristine, cytarabine, fludarabine and cladribine. Relapsed common-ALL were more in vitro resistant than common-ALL at first diagnosis to fludarabine and thioguanine. Lymphoblasts with TEL-AML1 rearrangement were more drug sensitive to L-asparaginase; lymphoblasts with MLL rearrangements were more sensitive to cytarabine and idarubicin. No differences in drug resistance were found for BCR-ABL lymphoblasts. No unambiguous relations were found between cell cycle parameters and cellular drug resistance. Conclusion: In vitro drug resistance in childhood ALL is related to biological features of lymphoblasts.