A likely endocrine control mechanism for sexual differentiation in size-graded populations of European sea bass (Dicentrarchus labrax) is proposed by evaluating the brain expression and pituitary content of two forms of gonadotropin-releasing hormone (GnRH), namely sea bream (sbGnRH) and salmon (sGnRH), the pituitary expression of one subtype of GnRH receptor (dlGnRH-R-2A) and the three gonadotropin (GtH) subunits, namely glycoprotein alpha (GPalpha), follicle-stimulating hormone beta (FSHbeta) and luteinizing hormone beta (LHbeta), as well as the pituitary and plasma LH levels between 50 and 300 days post-hatching (dph). Four gradings were conducted between 2 and 8 months after hatching, resulting in a population of large and small individuals, having 96.5% females (female-dominant population) and 69.2% males (male-dominant population), respectively, after the last grading. The onset of gonadal differentiation was different in the two sexes, and coincided with a peak of expression of sbGnRH or sGnRH. Furthermore, the expression of these GnRHs was correlated with the expression of dlGnRH-R-2A. Sex-related differences in the brain and pituitary content of sbGnRH were also found at the time of sexual differentiation. Moreover, the observed sexual dimorphism at the transcriptional or synthesis level of these GnRH forms suggests that a different neuro-hormonal regulation is operating according to sex. At the onset of sex differentiation, FSHbeta transcriptional activity reached maximal values, which were maintained until the completion of the process. The present study suggests a role for sbGnRH, sGnRH and the dlGnRH-R-2A during gonadal differentiation, possibly through enhancement of FSHbeta gene expression. In males, a different endocrine regulation seems to exist also during spermiogenesis and spermiation, when gene transcription, peptide synthesis and release of LH are of greater importance.