A number of variants of the wild-type (wt) estrogen receptor beta (ERbeta-1) coexist in a wide range of tissues. In the human these include, together with others, the expression of several isoforms (ERbeta-2-ERbeta-5) due to alternative splicing of exons encoding the carboxy terminus. In this study, we determined whether virally transformed cell cultures of human lens epithelial cells (HLE-B3) express both full length (or wt) and variant isoforms of ERbeta in comparison to normal secondary cultures of human lens epithelial cells (nHLE) and furthermore, identify the subcellular localization of the wtERbeta-1 and ERbeta isoform variants in HLE-B3 and nHLE cells, as well as from human breast adenocarcinoma cells (MCF-7) which provided a positive control. ERbeta isoform mRNA expression was evaluated by coupled RT-PCR. Subcellular localization of ERbeta isoforms was determined on formaldehyde-fixed, Saponin-permeabilized cells using conventional immunofluorescence techniques and affinity purified polyclonal antibodies specific for ERbeta-1 as well as to two of the truncated carboxy terminus isoforms (beta-2 and beta-5). Total RNA was extracted from HLE-B3 and nHLE cells and lens tissue, as well as from human breast adenocarcinoma cells (MCF-7) and subjected to RT-PCR using specific estrogen receptor primers intended to distinguish ERbeta-1-ERbeta-5 mRNA. The PCR products corresponded to wtERbeta-1 as well as to the isoform variants beta-2 and beta-5. The proportional distribution of wtERbeta-1, beta-2 and beta-5 PCR products differed between the normal lens epithelial cells and the SV-40 transformed lens epithelial cell line; the nHLE being similar to lens tissue with respect to relative expression of ERbeta isoform cDNAs. Confocal microscopy and immunofluorescence revealed ERbeta-2 was distributed throughout the cytosol and was associated with the nucleus of all cells examined, although sporadic immunostaining was observed with the nuclei of MCF-7. Prominent immunostaining of ERbeta-1 appeared in the mitochondria (along with weaker staining in the nucleus) of all cell types as authenticated by co-localization with Mitotrack-633. ERbeta-5 immunostaining was diffuse in the cytosol and also associated with the nuclei of all cell types. The differential subcellular partitioning of ERbeta-1 to the mitochondria and ERbeta-2 to the nucleus suggests a new aspect of regulation and function of the estrogen signalling system.