OBJECTIVE Deficit of the testosterone converting enzyme 17-beta-hydroxysteroid dehydrogenase (17beta-HSD) has been shown to be responsible for male pseudohermaphroditism (MPH). We analysed the gene encoding 17beta-HSD type 3 (17beta-HSD3) in a patient with MPH. METHODS We studied a 46, XY new-born diagnosed as having MPH. The child also had other congenital disorders, including a giant omphalocele and Fallot's tetralogy, and died of post-surgical complications at age 4.5 months. Basal hormonal levels, and after human chorionic gonadotrophin stimulation, suggested a deficiency in 17beta-HSD as the biochemical defect underlying this MPH. PCR amplification and subsequent sequencing of all coding exons of the 17beta-HSD3 gene were performed on genomic DNA from the patient and both parents. Messenger RNA was extracted from the patient's testis and 17beta-HSD3 cDNA was synthesized, PCR amplified and sequenced. RESULTS Sequencing revealed the presence of a homozygous missense mutation (R80W) in exon 3 of the 17beta-HSD3 gene, which was also present in single doses in both parents, in accordance with the recessive inheritance of the defect. No other mutation was found, and cDNA sequencing confirmed correct synthesis and processing of 17beta-HSD3 mRNA. CONCLUSIONS Confirming the abnormal delta4-androstenedione/testosterone ratios that suggested 17beta-HSD deficiency, a homozygous missense mutation in the gene coding for this enzyme was identified in the patient with MPH. This study adds further genetic evidence to the role of 17beta-HSD3 in male sexual development. There is no evidence supporting the association of this mutation in 17beta-HSD3 with the congenital malformations other than MPH present in the child.