Resistance to glucocorticoids remains one of the main obstacles in therapy of childhood acute lymphoblastic leukemia (ALL). The aim of the study was the analysis of relationship between ex vivo drug resistance of prednisolone and dexamethasone and exposure to these drugs in childhood ALL, with respect to risk factor analysis. Ex vivo resistance to both glucocorticoids was compared to maximum drug concentration achievable in body fluids, calculated in mathematical model. Drug resistance to vincristine and L-asparaginase, expression of multidrug resistance and apoptosis proteins was also determined. Concentration of both glucocorticoids in extracellular fluid was higher than drug resistance in the following groups of patients: in initial ALL patients, in patients staying in remission during follow-up, and in prednisolone good responders. Factors significant by multivariate analysis were early bone marrow response by day 15 and concentration of prednisolone higher than ex vivo prednisolone resistance. For initial ALL patients with determined response to initial prednisolone monotherapy, factors significant by univariate analysis were early bone marrow response, and exposure to glucocorticoids higher than ex vivo resistance to these drugs. No factor was significant by multivariate analysis in this group. Risk factor analysis showed that concentration of prednisolone and dexamethasone higher than respective ex vivo drug resistance, is a strongest prognostic factor in childhood ALL.