This study investigates the regulation of prostaglandin (PG) synthesis in the ovaries of sexually-mature zebrafish (Danio rerio). We examined the ovarian expression of genes within the arachidonic acid (AA) pathway, and the ovarian levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), 17beta-estradiol (E(2)), and PGF(2alpha) in spawning and nonspawning fish during the ovulatory cycle. Real-time RT-PCR analysis revealed that the expression levels of cytosolic phospholipase A(2) (cpla2) and cyclooxygenases (COX)-2 (ptgs2) in ovarian fragments and in isolated full-grown follicles of spawning fish were highest at 6:00 when ovulation was expected to occur. In nonspawning fish, cpla2 expression levels declined over time while ptgs2 expression displayed the same temporal pattern as in spawning fish. Elevated levels of 17alpha,20beta-P in the spawning fish occurred at 3:30, but there were no changes in the nonspawning fish. In other studies conducted to investigate the hormonal regulation of AA pathway genes, fish exposed via the water for 24 or 96 hr to 17alpha,20beta-P or E(2) exhibited reduced ovarian expression levels of COX-1 (ptgs1) and PG E synthase-2 (ptgsl), and E(2) reduced the expression of cpla2. Injection of human chorionic gonadotropin (hCG) (100 IU) led to increased expression levels of cpla2 and ptgs2 at 2 and 18 hr post-treatment, but consistently reduced ptgs1 and ptgsl expression. In these fish, ovarian levels of 17alpha,20beta-P were elevated at all time points and PGF(2alpha) levels in the hCG-treated group were significantly higher than the control fish at 18 hr. Collectively, these in vivo results suggest that gonadotropins and steroids are involved in the regulation of the AA pathway in ovarian follicles of zebrafish.