In the course of profiling alterations of gene expression in the male reproductive system induced by anti-androgenic agents, 28 genes expressed in the testis or pituitary of adult rats were examined shortly after subacute administration of the well-known anti-androgen, flutamide (FM). FM (25 mg/kg/day) was orally administered to male rats for six days. On day 8 (D8) after the first dose of FM, intratesticular testosterone (T) levels had dramatically increased, but daily sperm production on D36 was significantly decreased. The mRNA levels of testicular and pituitary genes on D8 were measured by semiquantitative RT-PCR. Among the six testicular steroidogenic enzyme genes, the mRNAs of the P450 side chain cleavage, P450 17 alpha/C(17-20) lyase, and 3beta-hydroxysteroid dehydrogenase type I (3betaHSD) genes significantly increased, whereas 17beta-hydroxysteroid dehydrogenase type III slightly decreased. Among the three steroid receptors examined, androgen receptor (AR) and glucocorticoid receptor (GR) mRNAs were significantly down-regulated (29% and 35%, respectively) in the testis, but there was no change in estrogen receptor alpha. There were no clear changes in expression of the gonadotropin receptors and Sertoli cell specific genes, but a slight increase was observed in expression of the lactose dehydrogenase-c mRNA, a germ cell specific gene. Among the three immediate early genes, c-myc mRNA was increased approximately 1.4-fold. In the pituitary, on the other hand, mRNAs for LHbeta and FSHbeta subunits and gonadotropin releasing hormone receptor had increased significantly. These results show that subacute FM administration first affected hypothalamus/pituitary hormone gene expression, then altered gonadotropin secretion, and subsequently induced over-expression of testicular steroidogenic enzyme genes. However, the significant up-regulation of 3betaHSD and down-regulation of AR mRNAs, despite the higher level of intratesticular T, might be explained by an antagonistic action of hydroxyflutamide retained in the testis. The profiles of alterations in gene expression observed will provide important information for the screening of adult male animals for anti-androgenic chemicals.