The steroid hormone 5 alpha-dihydrotestosterone (DHT) is one of the most physiologically important androgens in male vertebrates, with the exception of teleost fish, in which it is generally assumed that DHT does not play any major physiological role. However, this assumption is challenged by the fact that all the components involved in DHT biosynthesis and action are present and evolutionary conserved in teleost fish. In fact, testosterone (T) is converted into DHT by two isoforms of the enzyme steroid-5-alpha-reductase (5 alpha R), and both 5 alpha Rs gene expression and enzymatic activity have been detected in several tissues of different teleost species, which also have an androgen receptor with high binding affinity to DHT. This body of evidence strongly suggest that DHT is synthesised by teleost fish. We investigated this hypothesis using the cyprinid fathead minnow (Pimephales promelas) as the experimental model. The study of the evolutionary and functional conservation of 5 alpha Rs in teleost fish was used to support the experimental approach, based on an ultrasensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method to identify and measure simultaneously T and DHT in fathead minnow biological fluids and tissues. The analyses were performed using plasma samples collected from both male and female adult fish and samples of testicular tissue collected from sexually mature males. Both T and DHT were identified and quantified in all the samples analysed, and in particular, the high concentrations of DHT quantified in the testes suggested that these organs are a likely site of synthesis of DHT in the teleost fathead minnow, as they are in mammals. These results may represent the basis for future studies aimed at elucidating the physiological role, if any, of DHT in teleost fish. (C) 2013 Elsevier Inc. All rights reserved.