Gonad development in fish, as in mammals, is regulated by two gonadotropins (GTHs), FSH and LH. The function of LH in fish has been clearly established; however, the function(s) of FSH is less certain. The lack of specific and sensitive assays to quantify FSH and its α and β subunits has hindered studies to assess physiological function. In this study, gel filtration chromatography, ion exchange chromatography, and HPLC were employed to purify FSH and its subunits from pituitary glands of rainbow trout (Oncorhynchus mykiss), and the identities of the isolates were confirmed by amino acid analysis. Polyclonal antibodies were raised against the free GTHα2 and free FSHβ subunits to develop specific RIAs. The sensitivities of the intact FSH, GTHα2, and FSHβ assays were 1 ng/ml, 0.2 ng/ml, and 0.1 ng/ml, respectively, and the cross-reaction of these molecules with each other and with intact LH in the heterologous assays was <10.4% throughout. Pituitary and plasma samples diluted in parallel with the standards in all three assays and spiked sample recoveries were >90% throughout. Measurement of plasma and pituitary concentrations of intact FSH in female rainbow trout confirmed the established seasonal profiles. Concentrations of free GTHα2 subunit were elevated both in the plasma and in the pituitary in females at ovulation (maximum concentrations: 34.93 ± 6.3 ng/ml in plasma; 37.63 ± 5.79 μg/pituitary). In both the plasma and the pituitary, free FSHβ subunit was present throughout the reproductive cycle but at very low concentrations when compared with both free GTHα2 and intact FSH. The presence of free GTHα2 subunit in the plasma similarly occurs in mammals, but its functional significance in fish has yet to be established.