4-Hydroxy tamoxifen (OHT) and trilostane interact differently with the oestrogen receptor (ER). OHT is a competitive inhibitor whereas trilostane has direct, but non-competitive effects on ER. This study compared the effects of OHT and trilostane, in the presence of 17beta-oestradiol (E2) on gene expression in MCF-7 breast cancer cells using microarrays each representing nearly 20,000 human genes. Striking differences between the sets of genes affected by these two drugs were observed. Both OHT and trilostane affected transcription of genes involved in cell cycle regulation, cell adhesion and matrix formation, however, only 12.5% of trilostane down-regulated genes and 9.2% of up-regulated genes were similarly regulated by OHT. A selective up-regulation of ERbeta by trilostane, but not OHT, was observed and confirmed by qRT-PCR. Similar up-regulation of this gene by trilostane was observed in the uterus of trilostane-treated (4 mg/kg for 7 days) rats, in which ERbeta mRNA (3-fold) and ERbeta protein expression (10-fold) were both increased. These data show that OHT and trilostane regulate the expression of different sets of genes, reflecting their different modes of interaction with ER. Trilostane-specific up-regulation of ERbeta could explain its positive benefit rates in acquired tamoxifen resistance.