In the tilapia, Oreochromis niloticus, sex is determined by genetic factors (XX/XY) but temperature can also influence the gonadal sex differentiation. Elevated temperatures of 35 degreesC can generate functional male phenotypes if applied before and during sexual differentiation. The genes and mechanisms by which temperature acts on the cascade leading to sex differentiation have been investigated. Two strategies have been followed: 1) Search for novel genes by differential display, and 2) Expression studies of candidate genes. Genetically all-female and all-male progenies were reared at 27 degreesC (natural temperature) and at 35 degreesC (masculinizing treatment) and gonads dissected. Using differential display, we isolated a 300 bp cDNA (MM20C) from temperature-masculinized females. Virtual northern analysis revealed a 1.2 kb transcript in 35 degreesC treated females and males, but hardly any expression in natural females (27 degreesC). Semi-quantitative RT-PCR established a several-fold increase in MM20C expression in 35 degreesC masculinized fry. Elevated expression was observed in natural males (27 degreesC) with higher levels detected in those reared at 35 degreesC. Furthermore, we have analyzed as a candidate gene the P450 11 beta -hydroxylase, an important androgen steroidogenic enzyme. Low levels of expression were found in natural males. This coincides with low concentrations of 11 ketotestosterone in the gonads before and during gonadal sex differentiation. Higher expression levels of 11 beta -hydroxylase were detected in male gonads at 35 degreesC but levels in phenotypic males were similar to those found for natural females. Previous results reported that expression of aromatase is repressed by masculinizing treatments. Our study demonstrated that masculinizing-temperature can also stimulate the expression of other gene(s).