The EVI-1 gene was originally detected as an ectopic viral insertion site and encodes a nuclear zinc finger DNA-binding protein. Previous studies showed restricted EVI-1 RNA or protein expression during ontogeny; in a kidney and an endometrial carcinoma cell line; and in normal murine oocytes and kidney cells. EVI-1 expression was also detected in a subset of acute myeloid leukaemias (AMLs) and myelodysplasia. Because EVI-1 is expressed in the urogenital tract during development, we examined ovarian cancers and normal ovaries for EVI-1 RNA expression using reverse transcription polymerase chain reaction (RT-PCR) and RNAase protection. Chromosome abnormalities were examined using karyotypes and whole chromosome 3 and 3q26 fluorescence in situ hybridisation (FISH). RNA from six primary ovarian tumours, five normal ovaries and 47 tumour cell lines (25 ovarian, seven melanoma, three prostate, seven breast and one each of bladder, endometrial, lung, epidermoid and histiocytic lymphoma) was studied. Five of six primary ovarian tumours, three of five normal ovaries and 22 of 25 ovarian cell lines expressed EVI-1 RNA. A variety of other non-haematological cancers also expressed EVI-1 RNA. Immunostaining of ovarian cancer cell lines revealed nuclear EVI-1 protein. In contrast, normal ovary stained primarily within oocytes and faintly in stroma. Primary ovarian tumours showed nuclear and intense, diffuse cytoplasmic staining. Quantitation of EVI-1 RNA, performed using RNAase protection, showed ovarian carcinoma cells expressed 0 to 40 times the EVI-1 RNA in normal ovary, and 0-6 times the levels in leukaemia cell lines. Southern analyses of ovarian carcinoma cell lines showed no amplification or rearrangements involving EVI-1. In some acute leukaemias, activation of EVI-1 transcription is associated with translocations involving 3q26, the site of the EVI-1 gene. Ovarian carcinoma karyotypes showed one line with quadruplication 3(q24q27), but no other clonal structural rearrangements involving 3q26. However, whole chromsome 3 and 3q26 FISH performed on lines with high EVI-1 expression showed translocations involving chromosome 3q26. EVI-1 is overexpressed in ovarian cancer compared with normal ovaries, suggesting a role for EVI-1 in solid tumour carcinogenesis or progression. Mechanisms underlying EVI-1 overexpression remain unclear, but may include rearrangements involving chromosome 3q26.