Thyroxine (T4) level in the blood of newly hatched cockerels were measured at different times after the injection of bovine thyrotropin. A linear response to increasing doses of mammalian thyrotropin was seen when a two-injection protocol was used with a fifteen-hour interval between injections. Blood T4 levels peaked 5 hr after the second injection and declined thereafter. This response was shown to be highly specific for thyrotropin from both mammalian and avian sources; gonadotropins, prolactin, and growth hormone had negligible activity, although the last two hormones were able to synergise with thyrotropin under certain circumstances to augment the response. Experiments conducted at different times of day indicated that diurnal fluctuations in the response of the thyroid to exogenous thyrotropins may exist. As a result, animals were injected and bled at the same time of day in all subsequent experiments. Under these circumstances, in vivo thyroxine release in cockerels appears to be precise, simple, and sensitive bioassay for thyrotropin. This bioassay can be used to demonstrate that thyrotropin purified from ostrich pituitaries is distinct from gonadotropin and active in an avian species.