Measurements of plasma sodium levels, pituitary prolactin content, and detailed cytological studies of the pituitary prolactin cells were made on Poecilia latipinna adapted to different salinities and during adaptation to freshwater and 1 3 seawater. The prolactin band was identified in polyacrylamide gel disc electrophoregrams of pituitary homogenates. The optical density of the stained band was shown to be a measure of the amount of prolactin in the original homogenate. Plasma sodium is lowest in fish adapted to freshwater (FW), higher in 1 3 seawater ( 1 3 SW) and highest in seawater (SW). After transfer from 1 3 SW to FW, sodium falls during the first 18 hr, then rises to the normal FW range by 72 hr. After transfer from FW to 1 3 SW, sodium reaches 1 3 SW levels by 24 hr. Pituitary prolactin content is 6 times greater in FW than in SW, with intermediate levels in 1 3 SW. After transfer from 1 3 SW to FW prolactin content falls during the first 18 hr and then rises to FW levels by 8 days. The content remains high during the first 24 hr after transfer from FW to 1 3 SW. Content of putative growth hormone does not change with salinity. Cytological data show the prolactin cells to be more numerous and more active in FW than in SW; in 1 3 SW they are only slightly more active than in SW. After transfer from 1 3 SW to FW, cytological indices show that prolactin synthesis progressively increases, with loss of storage granules during the first 30 hr and return of granulation by 72 hr. The synthetic rate appears to be reduced 24 hr after transfer from FW to 1 3 SW. Considering all the data, it is concluded that in fish adapted to their surrounding medium, prolactin secretion rate is inversely proportional to salinity and directly proportional to pituitary prolactin content. During adaptation to new salinities, however, prolactin content and secretion rate are temporarily independent. Prolactin content in all circumstances closely parallels the granulation and size of the prolactin cells. The findings are discussed in relation to the known osmoregulatory effects of mammalian prolactin in teleosts, and are taken as confirming the physiological role of endogenous prolactin in adapting sodium metabolism to external salinity in P. latipinna.