Under physiological conditions, PRL synthesis and secretion are predominantly under negative control by dopamine acting through dopamine D2 receptors present in the pituitary lactotroph cells. To investigate the role of D4 receptors in the regulation of PRL synthesis and secretion, we stably transfected the human D4 receptor complementary DNA into the somatomammotrophic cell line GH4C1. The pharmacological characteristics of D4 expressed in GH4C1 were in close agreement with previous D4 receptor studies in Chinese hamster ovary and COS-7 cells. In GH4C1 cells, activation of D4 receptor variants (D4.2, D4.4, and D4.7) resulted in a similar level of reduction in forskolin- and vasoactive intestinal peptide (VIP)-stimulated cAMP levels (33% and 50%, respectively). In addition, the forskolin-stimulated activity of cAMP response elements fused to the VIP promoter driving the lacZ reporter gene could be blocked by D4 activation. However, quinpirole treatment had a minimal effect on transiently expressed luciferase reporter gene driven by a proximal PRL promoter in one of the D4-expressing cell lines. In contrast, the dopamine D2short receptor expressing GH4ZR7 cells treated with quinpirole displayed a significant decrease (51.3 +/- 4.1%) in PRL promoter activity. VIP-stimulated PRL release was not affected by D4 receptor activation, whereas in GH4ZR7 cells, a significant decrease in VIP-stimulated PRL levels was observed. Neither PRL promoter activity nor PRL secretion levels were affected in control untransfected GH4C1 cells. From this study it appears that although the D4 receptor may be expressed in the anterior pituitary, it does not have a major effect on PRL promoter activity or PRL secretion in GH4C1 cells despite its ability to reduce cAMP production. This might explain why D4- over D2-preferring antipsychotics such as clozapine do not cause hyperprolactinemia.