Recent advances towards the production of all-female populations of brook trout Salvelinus fontinalis have shown that methyltestosterone treatments applied after first feeding were not efficient in inducing masculinisation in neomales. A process to create all-female populations of brook trout using methyltestosterone was determined from 4 experiments. Different protocols of masculinisation by oral and/or hormonal immersion treatments were tested and the sex ratio of progeny produced from the cross of sex-reversed gynogenetic males with normal females was checked. No sexual dimorphism for growth was recorded, at least until two years of age. Masculinisation trials using oral treatments failed to produce any reversal of sex. Only the application of four weekly immersions beginning one week before hatching, combined with an oral treatment, provided 100% masculinisation. Gynogenetic sex-reversed males crossed with normal females produced 100% females, demonstrating the efficiency of this protocol in reversing the sex of brook trout. This result also highlights an earlier and longer period of sex lability, compared with most other Salmonids, and the need to initiate immersion treatments a long time before the initiation of gonadal differentiation.