Mammary gland ion transport is essential for lactation and is regulated by prolactin and glucocorticoids. This study delineates the roles of prolactin receptors (PRLR) and long-term prolactin and dexamethasone (P-D)-mediation of [Ca(2+)](i) and Cl(-) transport in HC-11 cells. P-D (24 h) suppressed ATP-induced [Ca(2+)](i). This may be due to decreased Ca(2+) entry since P-D decreased transient receptor potential channel 3 (TRPC3) but not secretory pathway Ca(2+)-ATPase 2 (SPCA2) mRNA. ATP increased Cl(-) transport, measured by iodide (I(-)) efflux, in control and P-D-treated cells. P-D enhanced I(-) efflux response to cAMP secretagogues without altering Cl(-) channels or NKCC cotransporter expression. HC-11 cells contain only the long form of PRLR (PRLR-L). Since the short isoform, PRLR-S, is mammopoietic, we determined if transfecting PRLR-S (rs) altered PRLR-L-mediated Ca(2+) and Cl(-) transport. Untreated rs cells showed an attenuated [Ca(2+)](i) response to ATP with no further response to P-D, in contrast to vector-transfected (vtc) controls. P-D inhibited TRPC3 in rs and vtc cells but increased SPCA2 only in rs cells. As in wild-type, cAMP-stimulated Cl(-) transport, in P-D-treated vtc and rs cells. In summary, 24 h P-D acts via PRLR-L to attenuate ATP-induced [Ca(2+)](i) and increase cAMP-activated Cl(-) transport. PRLR-S fine-tunes these responses underscoring its mammopoietic action.