Glutathione S-transferases are involved in the conjugation of a number of human carcinogens. The frequencies of the deletion alleles coding for GSTM1, and GSTT1, related to deficient conjugation of xenobiotics, as well as a recently reported variant in the exon 5 of GSTP1 were investigated in this study. A multiplex polymerase chain reaction based method for a rapid and high throughput genotype analysis of all three GSTM1, GSTT1 and GSTP1 genes in a single tube was developed. Leukocyte DNA from two hundred and thirty-nine (n = 239) breast cancer patients were genotyped. Tumors from a subset of these breast cancer patients (n = 131) have previously been investigated for mutations in the TP53 gene, levels of p53 protein accumulation and loss of heterozygosity at several loci on chromosome 17. When genetic alterations in the tumors were analyzed with respect to glutathione S-transferase genotypes, a significantly higher proportion of the patients with a G allele (GG + AG) of the GSTP1 had loss of heterozygosity at the TP53 gene locus mapping to 17p, compared with non-G allele carriers (74% versus 29%) (P = 0.018). The patients carrying the G allele of GSTP1 also had more frequently mutations in the TP53 gene in their tumor (38%), compared with patients with the AA genotype (21%) (P = 0.055). G allele carriers had predominantly deletion or transversion mutations in the TP53 gene (5 of 7 and 5 of 6 respectively). A higher frequency of the G allele carriers was observed among patients with negative lymph node status (P = 0.0004). A higher proportion of the patients with positive lymph node status at the time of diagnosis had a combined GSTM1 null/GSTT1 null genotype (P = 0.05). Patients who were homozygous for the deleted GSTM1 allele were found to have a significantly shorter overall survival (P = 0.036).