In oviparous species, in addition to a full-length estrogen receptor alpha (ERα), another ERα isoform lacking the A domain and exhibiting a ligand-independent transactivation function has been consistently reported. Although both isoforms are expressed in the liver, their respective sites of expression in other potential target tissues are unknown. In contrast to the situation in Xenopus and chicken, the two isoforms of rainbow trout (Oncorhynchus mykiss) are generated from two classes of transcripts with different 5′ untranslated sequences issued from the same gene by alternative splicing and promoter usage. The aim of this study was to take advantage of the unique organization of the rainbow trout ERα gene to investigate the tissue distribution of these two messenger species along the reproductive axis of female trout. The S1 nuclease assay and in situ hybridization were used, with probes specific for each of the transcripts. Reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the isoforms also was performed. The data indicated that the full-length ERα is expressed in liver, brain, pituitary, and ovary, whereas expression of the isoform with the truncated A domain is restricted to the liver, demonstrating a tissue-specific expression of these two ERα isoforms. The presence of a short liver-specific isoform in oviparous species suggests its role in the development and/or maintenance of the unique function of the liver in the vitellogenesis process.