A sensitive semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was performed to evaluate estrogen receptor-α (ER-α) mRNA expression in prostate cancer cell lines. We demonstrated the presence of wild-type ER-α (wt ER-α) and five ER-α variants, designated ER-αA, B, C, D, and E. Unlike ER-αA and D, ER-αB, C, and E were not previously reported in normal or cancerous mammalian cells. DNA sequencing analysis of these ER-α variants revealed the genetic changes to be either in-frame or out-of-frame deletions. The expression of each ER-α variant differs significantly depending on the androgen responsiveness, tumorigenic and metastatic potentials of each prostate cancer cell line. The potential functional significance of ER-α variants was assessed in yeast two-hybrid and ERE promoter-reporter mammalian transcription assay systems. The results of these studies indicated that none of the ER-α variants can form homo- or heterodimers either with wt ER-α or among themselves in vivo, and that these ER-α variants have no demonstrable transcriptional or dominant-negative activity, as assessed in vitro.