Prognostic signatures in breast cancer derived from microarray expression profiling have been reported by two independent groups. These signatures, however, have not been validated in external studies, making clinical application problematic. We performed microarray expression profiling of 135 early-stage tumors, from a cohort representative of the demographics of breast cancer. Using a recently proposed semisupervised method, we identified a prognostic signature of 70 genes that significantly correlated with survival (hazard ratio (HR): 5.97, 95% confidence interval: 3.0-11.9, P = 2.7e-07). In multivariate analysis, the signature performed independently of other standard prognostic classifiers such as the Nottingham Prognostic Index and the 'Adjuvant!' software. Using two different prognostic classification schemes and measures, nearest centroid (HR) and risk ordering (D-index), the 70-gene classifier was also found to be prognostic in two independent external data sets. Overall, the 70-gene set was prognostic in our study and the two external studies which collectively include 715 patients. In contrast, we found that the two previously described prognostic gene sets performed less optimally in external validation. Finally, a common prognostic module of 29 genes that associated with survival in both our cohort and the two external data sets was identified. In spite of these results, further studies that profile larger cohorts using a single microarray platform, will be needed before prospective clinical use of molecular classifiers can be contemplated.