Changes in steroid hormone levels in the serum and ovarian fluid were studied during overripening in goldfish. Ovulated eggs retained in the ovarian cavity become overripe at around 12 h after ovulation and completely overripe 24 h after. Blood and ovarian fluid were taken at 0, 3, 6, 12, 18, and 24 h after ovulation. Estradiol-17ß (E 2) , testosterone (T), progesterone (P) and 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-P) in the serum were extracted directly with a solvent while those in the ovarian fluid were separated by HPLC before radioimmunoassay. Both serum and ovarian fluid P showed a highly significant decline at 18 h with a further decline at 24 h; P levels were higher in the ovarian fluid. Serum 17α,20ß-P showed a progressive and more rapid decline, decreasing significantly at 12 h with further decreases at 18 h and 24 h; the level was five-fold lower at 24 h compared to the 0 h level. Serum T increased significantly at 3 h which was maintained until 18 h, when it declined to 0 h level. No significant changes in E2 were observed in the serum, except for a significant difference between 6 and 24 h. There were no significant changes in E2, T and 17α,20ß-P in the ovarian fluid. Of the four steroids measured, only 17α,20ß-P and P showed changes which bear some correlation with the time course of overripening. The declines in the mean ratios of 17α,20ß-P/E2 in the serum and P/E2 in the ovarian fluid also appeared to have a good correlation with the time course of overripening. The postovulatory follicles (POFs) showed degenerative features which likewise correspond to the decline in P and 17α,20ß-P.