Drug resistance assays may be useful to identify drug interactions. For this purpose, we studied three drug combinations, each at 8-12 concentrations, with the MTT assay in acute lymphoblastic leukemia (ALL) samples from 34 children obtained at initial diagnosis. This resulted in a total of 518 comparisons between expected and observed leukemic cell survivals. The combinations prednisolone (PRD) with vincristine (VCR), PRD with mafosfamide (MAF), and PRD with daunorubicin (DNR) were tested without technical difficulties, and without an increased assay variation as compared to single drugs. We observed a marked heterogeneity in drug interactions between patients, between combinations, and between different concentrations within one specific combination. Between PRD+VCR, synergism was found in 46%, antagonism in 18%, and additivity in 36% of the 228 observations. Between PRD+MAF, synergism was found in 51%, antagonism in 20%, and additivity in 29% of the 140 observations. Between PRD+DNR, synergism was found in 35%, antagonism in 31%, and additivity in 34% of the 150 observations. PRD+VCR and PRD+MAF showed more often synergism than PRD+DNR, while antagonism was observed more frequently between PRD+DNR (p < 0.05). However, the magnitude of antagonism was not much different between the three drug combinations, nor was there a significant antagonistic interaction in any of the drug combinations tested, if all samples were considered together. We conclude that the MTT assay can be used to study drug interactions in vitro in ALL samples. The type of interaction was different between patients, and depends on the drug combination and concentrations. The combinations PRD+VCR and PRD+MAF generally showed additive and even synergistic interactions. The cytotoxicity of PRD+DNR was generally not markedly higher than that of the most active single drug.