Detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) predicts outcome. Previous studies were invariably based on relative quantification and did not investigate sample-inherent parameters that influence test accuracy, which makes comparisons and clinical conclusions cumbersome. Hence, we conducted a prospective, population-based MRD study in 108 sequentially recruited children with ALL uniformly treated with the ALL-Berlin-Frankfurt-Münster (ALL-BFM) 95 protocol in Austria (median follow-up of 40 months). Using sensitive, limited antibody panel flow cytometry applicable to 97% of patients, we investigated 329 bone marrow samples from 4 treatment time points. MRD was quantified by blast percentages among nucleated cells (NCs) and by absolute counts (per microliter). Covariables such as NC count, normal B cells, and an estimate of the test sensitivity were also recorded. Presence and distinct levels of MRD correlated with a high probability of early relapse at each of the time points studied. Sequential monitoring at day 33 and week 12 was most useful for predicting outcome independently from clinical risk groups: patients with persistent disease (> or =1 blast/microL) had a 100% probability of relapse, compared to 6% in all others. Absolute MRD quantification was more appropriate than relative, due to considerable variations in total NC counts between samples. Regeneration of normal immature B cells after periods of rest from treatment limited the test sensitivity. In conclusion, MRD detection by flow cytometry is a strong and independent outcome indicator in childhood ALL. Standardization regarding absolute quantification on the basis of NCs and assessment during periods of continuous treatment promise to increase the accuracy, simplicity, and cost efficiency of the approach.