The ovulation-inducing property of androgens in the laying hen was investigated. In a first experiment, four different androgens were injected subcutaneously into single-comb White Leghorn hens on the day of the last oviposition of a sequence. The hens were killed 10 h later and examined for the presence of an ovum in the oviduct. Testosterone induced ovulation in accordance to the dose injected (median effective dose, 966 +/- 193 microgram/hen) but the responses to 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were not dose-related. The effect of 4-androstene-3, 17-dione was more like that of progesterone since it induced ovulation 2 h earlier than the three other androgens. The physiological significance of the ovulation response to an injection of testosterone was examined in more detail in experiment 2. Seven out of ten hens which were injected with 1 mg testosterone/kg body weight ovulated within 10 h after the injection. Blood samples were taken at hourly intervals and the concentrations of testosterone and progesterone were determined by radioimmunoassay. An injection of testosterone produced an increase in the concentration of testosterone in plasma which was considerably greater and occurred earlier than the preovulatory increase of testosterone in the control birds. The increase in the concentration of progesterone in the hens injected with testosterone was similar in magnitude but occurred earlier than the spontaneous preovulatory increase of progesterone in the control hens. The possible physiological role of testosterone in the ovulation cycle is discussed.