The effects of prolactin and vasopressin on the regulation of amniotic fluid (AF) volume and its Na⁺ concentration Na⁺ through the membrane surrounding the AF during increase in AF volume due to fetal urination were studied. About 70% of AF volume was replaced with normal isotonic saline solution. Isotonic saline solution (0.5 ml) containing Censored and LiCl was introduced into each amniotic sac. Vasopressin (25 ng/ml) or prolactin (1 mg/ml) of AF was then injected into experimental amniotic sac. The concentrations of Congored, Li⁺, and Na⁺ were measured at 30 and 60 min intervals after injection. Af samples with decreased Censored concentration ([CR]) during the period of 30 - 60 min were analyzed. The percentage change of [Na⁺] and the rate of Li⁺ movement during this period were calculated, and the effects of vasopressin and prolactin on them were evaluated. Fellowing results were obtained: 1. The rate of reduction of [CR] in the AF was retarded by vasopressin or prolactin injection. 2. The rate of reduction of [Li⁺] in the AF was also retarded by vasopressin or prolactin injection. 3. The rate of reduction of [Li⁺] in the AF was less retarded by vasopressin than that of [CR]. 4. [Na⁺] changed to approach to the normal level, but this was markedly retarded by prolactin injection. 5. Direction of Li⁺ movement was correlated with the change in [Na⁺] but it always moved out of the amniotic sac even when the [Na⁺] increased in vasopressin injected AF. From the above results, it is suggested that vasopressin in the AF triggers the fetus to urinate, and then the membranes surrounding the AF regulate osmolarity by efflux of Na⁺. We suggest that prolactin facilitates water outflow across the amniotic membrane during increase in AF volume, in contrast to a constant volume, whereas regulation of [Na⁺] is partly restricted by prolactin.