A cloned thymosin beta-10 cDNA and a synthetic oligonucleotide specific for the thymosin beta-4 gene were used to study the in vivo expression of these two genes in the immature rat ovary in response to exogenously administered gonadotropins. Despite the fact that both genes were co-expressed in the rat ovary, it became evident that they exhibit distinctly unique differential responses to in vivo hormonal challenge. Administration of pregnant mare's serum gonadotropin (PMSG) to immature rats provoked a pronounced stimulation of ovarian thymosin beta-10 expression, the maximal effect (2- to 4-fold) of which coincided with the time at which folliculogenesis was also maximally enhanced. In contrast, the transcriptional status of the thymosin beta-4 gene varied little in response to the PMSG. Administration of human chorionic gonadotropin (hCG) to PMSG-primed rats inhibited ovarian thymosin beta-10 but stimulated thymosin beta-4 gene expression. These findings suggest that despite a mutually high degree of homology, these two proteins may, under the influence of gonadotropins, play independent roles in normal ovarian function.