Release of prolactin from both normal pituitary cells and rat pituitary tumor (GH) cells is an osmotic process that is dependent upon chloride. The long term growth rate of GH-cells in medium in which chloride was exchanged with isethionate was completely normal, but, by 48 h, isethionate substitution resulted in a 70% decrease in the concentration of internal and secreted prolactin. Isethionate caused a much smaller reduction in growth hormone production (less than 20%). These results suggest that exchange of chloride with isethionate is inhibiting the synthesis of prolactin. Reduction of intracellular levels of prolactin in cells grown in isethionate-containing medium was evident by 30 h, and the level of prolactin was reduced 92% at 96 h. This reduction in the internal concentrations of prolactin was reversed when the cells were returned to normal medium containing chloride with a t1/2 of 48 h. Addition of epidermal growth factor and the calcium channel agonist BAY K 8644 to cells in medium containing chloride increased internal prolactin by 400%, and isethionate exchange reduced the response by 85%. To confirm that isethionate exchange was inhibiting the synthesis of prolactin, mRNA concentrations for prolactin and actin were determined. Both basal and hormone-stimulated levels of prolactin mRNA were reduced 70 to 90% by isethionate exchange, while actin mRNA levels did not change. To determine whether the effect of isethionate was at the level of gene transcription, GH-cells were transfected with a prolactin-chloramphenicol acetyltransferase fusion gene and chloramphenicol acetyltransferase expression was assessed using cells in chloride and isethionate-containing media. Both basal and hormone-stimulated synthesis of chloramphenicol acetyltransferase driven by the prolactin promoter was inhibited by isethionate exchange. These studies demonstrate that exchange of medium chloride with isethionate inhibits the synthesis of prolactin at the level of transcription.