The POU transcription factor Pit-1 activates the prolactin gene in pituitary lactotrophs and may integrate responses of the gene to external signals. To study the role of Pit-1 in dopaminergic inhibition of the prolactin gene, we transiently transfected Pit-1 and dopamine D2 receptor vectors into a series of heterologous cell lines and examined dopamine regulation of the prolactin gene promoter. Regulation was Pit-1-dependent in all cell lines tested. Moreover, dopamine responsiveness was cell type-specific: stimulatory in fibroblasts (COS-7) and muscle-type cells (P19/Me2SO-induced) and inhibitory in pancreatic endocrine (RIN, InR1-G9) and neural-like (P19/retinoic acid-induced) cells. Because dopaminergic responses in Pit-1-transfected RIN cells paralleled those in pituitary GH4 cells, the islet cell line was used to test for sequences in Pit-1 that mediate negative hormone signals. Dopamine responsiveness of the Pit-1 transactivation domain (residues 8-80) was examined using a chimeric LexA construct. LxPit-1, LxSp1, and Lx-glucocorticoid receptor fusions all activated basal transcription, but only LxPit-1 was regulated by dopamine. Regulatory responses of LxPit-1 and full-length Pit-1 were quantitatively similar. In addition, gain-of-function G alpha mutants that inhibit Pit-1-dependent promoters in GH4 cells also suppressed selectively Pit-1- or LxPit-1-dependent promoters in RIN cells. This demonstrates that Pit-1 can function as a specific target for distinct inhibitory G protein signals. Interestingly, Pit-1 sequences N-terminal to the DNA-binding POU domain appear to be sufficient in mediating regulation by these pathways.