Immunological cross-reactivities between rainbow trout GTH I and GTH II and their alpha and beta have been studied using highly purified rainbow trout gonadotropins and subunits and antibodies raised against beta subunits. From these observations radioimmunoassays have been developed for rainbow trout GTH I and GTH II. The GTH II RIA was highly specific and cross-reacted only with GTH II and its beta 1 subunits, with beta 2 being less potent than beta 1 in competing GTH II binding. There was no cross-reactivity with GTH I. Its sensitivity varied between 0.1 and 0.2 ng/ml, allowing GTH II measurement early in the reproductive cycle. Variations between and within assays were less than 10%. There was a lack of specificity of GTH I RIA (44% cross-reactivity with GTH II, when using labelled native GTH I). Reasons for this lack of sensitivity were studied. It cannot be attributed to beta subunits (less than 1.2% cross-reactivity). However, the cross-reactivity of alpha subunits was very important. This suggests that the presence of free alpha subunits in the medium can be responsible for the lack of specificity. Labelling native GTH I resulted in conformational change in molecular weight and dissociation of the hormone into subunits, whereas iodination did not induce GTH II dissociation. This dissociation can be avoided by labelling the stable form of GTH I. Using this radio-tracer, the specificity and the sensitivity of the assay were greatly improved (GTH II cross-reactivity was decreased to 3.7, mean sensitivity 0.87 +/- 0.072 ng/ml). The sensitivity of the assay diminished with ageing of labelled GTH I. The assay variation was 4.6% within an assay and 9.8% between assays. The use of labelled beta GTH I still increases the specificity (2.3% GTH II cross-reactivity), but with a 2.4-fold loss of sensitivity. In both GTH I and GTH II RIA plasma and spiked plasma with purified GTHs gave displacement curves parallel to standard. These assays were used to study pituitary responsiveness to a GnRH analogue in female rainbow trout prior to oocyte maturation. The effects of GnRH on GTH II secretion were confirmed. The peptide did not significantly stimulate GTH I secretion.