Pre-B-cell leukaemia transcription factors (PBX1, PBX2, PBX3 and PBX4) are part of the TALE (three amino acid loop extension) homeobox gene family. They regulate gene expression programs during development and steroidogenesis. In this study, we evaluated PBX expression in prostate cancer cell lines and tissue sections from radical prostatectomy of prostate cancer patients. We detected PBX1, PBX2, PBX3 and PBX4 at mRNA level in all prostate cell lines tested by using Real-Time RT-PCR. Furthermore, isoforms of PBX1 and PBX3 were observed at the protein level using Western analysis. Androgen down-regulated mRNA levels of PBX1 and PBX4. Hormonal regulation of PBX1 mRNA was also seen in human prostate tissue samples. Although no effects of androgens were observed on PBX3 mRNA level, androgens were shown to decrease the protein level of PBX3 in LNCaP. Immunohistochemical staining of PBX3 showed increased expression of PBX3 in malignant versus benign prostate tissue. Malignant samples were clearly positive for nuclear and cytoplasmic PBX3 staining in luminal cells, whereas benign samples were negative. Distinct nuclear staining in basal cells, however, were observed in bening areas. Up-regulation of PBX3 mRNA was also observed in tumour samples from one of three prostate cancer patients compared to paired malignant biopsy. Our observations showed that members of the PBX family were regulated by androgens through different mechanisms and indicated that PBX3 may play a role in prostate cancer progression.