Transcription of the prolactin gene is dynamically controlled by positive and negative hormone signals that target the regulatory promoter region. Based on the inducibility of prolactin gene expression by inhibitors of histone deacetylases, we examined the role of histone acetylation at the genomic prolactin promoter as a late step in transcriptional regulation. Chromatin immunoprecipitation analysis of GH4 cells revealed elevated levels of acetylated histones in the promoter and enhancer regions of the gene, compared with downstream intron sequences. Estradiol 17-beta stimulated histone H4 acetylation in the promoter region by 2 to 3-fold within 30 min. Dopamine inhibited histone H4 acetylation by 2-fold in 30 min, an effect mimicked by the MAPK kinase inhibitor U0126. In contrast, the synthetic glucocorticoid dexamethasone, which inhibits prolactin transcription, failed to alter histone acetylation over the same time frame. Association of transcription activator Pit-1 with the prolactin promoter was unchanged by hormone treatment. However, in response to dopamine, histone deacetylase HDAC2 and corepressor mSin3A were rapidly recruited to the prolactin promoter and association sustained above basal levels over a one hour period. Consistent with this corepressor function, depletion of endogenous mSin3A by siRNA was sufficient to enhance prolactin gene expression by 70%, comparable to the induction by the HDAC inhibitor, TSA. These studies demonstrate that dopamine D2 receptor activation and inhibition of ERK1/2 signaling lead to rapid deacetylation of histones at the genomic prolactin promoter. Recruitment of specific HDAC/ corepressor complexes may be an important mechanism for repression of target gene transcription by Gi/o-coupled receptors.