Current treatment protocols for childhood acute lymphoblastic leukemia always include thiopurine drugs such as 6-mercaptopurine (6-MP), which is administrated daily during maintenance therapy. Once 6-MP is an inactive prodrug, it needs to be activated to thioguanine nucleotides (TGNs) to exert cytotoxicity. However, 6-MP can also be methylated by the polymorphic enzyme thiopurine S-methyltransferase (TPMT), thus reducing TGNs formation. In order to assess the influence of TPMT polymorphism in ALL, we have used PCR/HCSGE (Horizontal Conformation-Sensitive Gel Electrophoresis) based methods to characterise molecularly a sample of 110 children with ALL. Four distinct alleles associated with TPMT deficiency (TPMT*3A, *3C, *2 and *8) were found in heterozygous individuals representing 11.8% of the sample. We have also compared several parameters related with ALL outcome between subsamples of children homozygous or heterozygous for TPMT. In the heterozygous group, 6-MP dosages were lower and the number of whole interruptions during treatment or interruptions due to toxicity as well as the number of relapses was higher compared to the other group. Despite our results do not reach statistical significance, this study emphasizes the importance to introduce the prospective analysis of TPMT genotype, prior to any ALL treatment.