The cure rate for acute lymphoblastic leukemia (ALL) may exceed 85%. Up to three years of maintenance therapy with weekly methotrexate (MTX) and daily 6-mercaptopurine is required. Compliance with maintenance is essential to eliminate the residual leukemic clone. We determined the compliance status of children with ALL treated at a University Hospital. The study had three phases: a direct structured interview, the search for lack of compliance documented in the clinical files, and measurement of serum MTX levels, in three random occasions, using a polarized fluorescent immunoassay. In 5 of 49 (10%) interviews, at least an episode of non-compliance was reported. In the clinical file, 8 of 49 (16.3%) patients referred skipping maintenance drugs. In 14 of 49 (28.6%) children, MTX was not present in serum in at least one measurement. Face-to-face interviews and clinical file notes were unreliable sources for monitoring compliance. Random determination of serum MTX levels should be given consideration for monitoring long-term compliance with maintenance therapy in children with ALL.