Established techniques of genetic manipulation were used to elucidate sex-determining mechanisms in the commercially important tilapia, Oreochromis niloticus. Analysis of sex ratios from single-pair matings of normal broodstock showed these to be heterogeneous, with an asymmetrical frequency distribution. Data were homogeneous, with the exclusion of a number of broods with sex ratios not significantly different from 3:1 (male:female), and further progeny testing revealed atypical female heterogamety in the parents of these broods. Analysis of sex ratios from complete diallele-type crosses using five males and five females demonstrated no association between male parent, female parent and progeny sex ratio. Sex ratios of gynogens (0:1) and triploids (1:1), and from progeny testing of sex-reversed males (0:1) and sex-reversed females (3:1), provide evidence for female homogamety in this species. Progeny testing of male gynogens derived from sex-reversed females demonstrated recombination between the centromere and the sex-determining locus (68.9%). Novel YY "supermales" were shown to be viable and produced all-male offspring. It was concluded that this species exhibits monofactorial, genotypic sex determination with male heterogamety. However, rare autosomal or environmental sex-modifying factors may account for occasional deviations from expected sex ratios.