The estrogen receptor gene gives rise to variant mRNAs, generated by alternative mRNA splicing, as well as the full-length mRNA containing eight coding exons. It has been postulated that one of these, the exon 5 variant, may be important in the development of hormone-independent and anti-estrogen-resistant breast cancer since it has the potential to encode a truncated receptor that retains the N-terminal activation domain AF-1, but lacks the hormone-binding domain. We have expressed the variant using an inducible promoter in estrogen receptor-positive MCF-7 cells and analyzed the effect of the variant protein on gene expression and cell growth. Inducible expression was validated using a specific antiserum that recognized a novel epitope on the exon 5 variant. The variant was able to stimulate transcription of a reporter gene in transiently transfected chicken embryo fibroblasts in the absence of hormone but showed weak constitutive activity when it was stably expressed in MCF-7 cells. The variant had no effect on the expression of the estrogen target genes, pS2, and the progesterone receptor. Finally, we analyzed whether the proliferation of MCF-7 cells was altered by the expression of the exon 5 variant and found that the stimulatory effects of estrogen and growth inhibitory effects of tamoxifen and ICI 182780 were unchanged. We therefore conclude that expression of the variant alone is not sufficient to give rise to hormone independence or tamoxifen resistance of breast cancers.