Plasma prolactin and TSH concentrations were measured by radioimmunoassay in lactating rats after exposure to ether fumes, suckling, treatment with nicotine or injection of synthetic TRH. Both 100 ng and 100 μg of TRH caused a significant elevation in plasma prolactin within 2 min after injection. Prolactin remained elevated for 30 min after administration of 100 μg. The response seen after injection of 100 ng of TRH was similar to the rise in plasma prolactin seen after treatment with nicotine or exposure toether. However, neither nicotine nor ether fumes caused an increase in plasma TSH. Suckling caused a marked increase in circulating levels of both prolactin and TSH but the rise in plasma prolactin preceded the rise in plasma TSH by more than 5 min. In contrast, TRH caused a rapid (within 2 min) in crease in circulating prolactin and TSH. Neither nicotine nor ether altered TSH release in response to TRH and nicotine did not block the rise in plasma prolactin after TRH. In cycling rats, plasma prolactin but not TSH rose on theafternoon of proestrus. Nicotine treatment delayedthe rise in prolactin and again was without effect on plasma TSH concentration. Exogenous TRH increased plasma prolactin and TSH concentrationearly on the afternoon of proestrus similar to the results seen in lactating rats. Thedata indicate that TRH can increase radioimmunoassayable plasma prolactin in addition to TSH inlactating and cycling rats but TRH alone is not the factor responsible for the rise in plasma prolactinseen after exposure to ether, suckling or treatment with nicotine nor for the prolactinsurge seen on the afternoon of proestrus. (Endocrinology94: 503, 1974)