We have investigated the effect of HER-2 overexpression on resistance to the aromatase inhibitor letrozole in MCF-7 breast cancer cells stably expressing cellular aromatase (MCF-7/CA). MCF-7/CA cells overexpressing HER-2 showed a >2-fold increase in estrogen receptor (ER) - mediated transcriptional reporter activity upon treatment with androstenedione compared with vector-only control MCF-7/CA cells. Cotreatment with letrozole did not abrogate androstenedione-induced transcription and cell proliferation in HER-2-overexpressing cells. Chromatin immunoprecipitation assays using cross-linked protein-DNA from MCF-7/CA/HER-2 cells indicated ligand-independent association of the ERα coactivators AIB-1 and CBP to the promoter region of the estrogen-responsive pS2 gene. Upon treatment with androstenedione, there were increased associations of AIB1 and CBP with the pS2 promoter in the HER-2-overexpressing compared with control MCF-7/CA cells. These results suggest that ligand-independent recruitment of coactivator complexes to estrogen-responsive promoters as a result of HER-2 overexpression may play a role in the development of letrozole resistance.