The extensive heterogeneity of breast cancer complicates the precise assessment of tumour aggressiveness, making therapeutic decisions difficult and treatments inappropriate in some cases. Consequently, the long-term metastasis-free survival rate of patients receiving adjuvant chemotherapy is only 60%. There is a genuine need to identify parameters that might accurately predict the effectiveness of this treatment for each patient. Using cDNA arrays, we profiled tumour samples from 55 women with poor-prognosis breast cancer treated with adjuvant anthracycline-based chemotherapy. Gene expression monitoring was applied to a set of about 1000 candidate cancer genes. Differences in expression profiles provided molecular evidence of the clinical heterogeneity of disease. First, we confirmed the capacity of a 23-gene predictor set, identified in a previous study, to distinguish between tumours associated with different survival. Second, using a refined gene set derived from the previous one, we distinguished, among the 55 clinically homogeneous tumours, three classes with significantly different clinical outcome: 5-year overall survival and metastasis-free survival rates were respectively 100% and 75% in the first class, 65% and 56% in the second and 40% and 20% in the third. This discrimination resulted from the differential expression of two clusters of genes encoding proteins with diverse functions, including the estrogen receptor (ER). Another finding was the identification of two ER-positive tumour subgroups with different survival. These results indicate that gene expression profiling can predict clinical outcome and lead to a more precise classification of breast tumours. Furthermore, the characterization of discriminator genes might accelerate the development of new specific and alternative therapies, allowing more rationally tailored treatments that are potentially more efficient and less toxic.