In fish, both gonadotropin (GtH)-I and -II are involved in the spermatogenic process, but the differential regulation of these hormones by GnRH is still poorly understood. To gain further insight into the GnRH regulation of GtH-I and -II gene expression in the male striped bass, we have developed and optimized a ribonuclease protection assay for the simultaneous measurement of all GtH subunit mRNAs in a single pituitary gland. The RNA extraction protocol enables the determination of GtH protein content in the same sample, thus enhancing the power of the method. Maturing striped bass males were injected intramuscularly with [D-Ala6,Pro9Net]-LHRH (GnRHa) and sampled at 6 and 24 h postinjection. The mRNA levels of the α subunit and GtH-IIβ increased after 6 h (4- and 6-fold, respectively), while the GtH- Iβ mRNA levels increased only 2-fold after 24 h. Interestingly, GnRHa stimulation caused a significant increase in β-actin mRNA levels. GnRHa treatment also resulted in a 2-fold decrease in pituitary GtH-II content, associated with a dramatic increase of plasma GtH-II levels from undetectable levels (< 0.2 ng/ml) to 13 ± 2 ng/ml after 6 h. These results demonstrate that both GtH-Iβ and -IIβ are expressed during striped bass spermatogenesis and that the two genes are subjected to differential regulation by GnRHa.