Background: Despite many published studies on ERβ, progress towards understanding its role in breast cancer remains slow. This is largely due to discordant data between mRNA and protein studies as well as failure to take into account the biologically distinct ERβ isoforms and their heterogeneous expression profile.Methods: We compared expression of ERβ, -2 and -5 genes in HB2 and MCF-7 breast cell lines, primary breast fibroblasts (n=5) and whole tissue and laser microdissected epithelial and stromal cells obtained from 25 human breast tumours.Results: Our study shows that the level of gene expression of ERβ isoforms depends on the cell population within a given tumour and varies dramatically in different cellular compartments. This has implications for gene expression analyses and could explain some of the contradictory data published to date, rendering “grind and bind” analyses of ERβ uninformative.Conclusions: With the technology now available, we suggest a more refined approach be adopted to help resolve some of the controversy surrounding ERβ.