The secretory cells of the anterior pituitary are regulated by hypothalamic and target endocrine hormones. In many cases, intracellular signaling after ligand binding to cell surface receptors is mediated by heterotrimeric G proteins. In this study, cells from the rat pituitary and two pituitary cell lines (GH3 and AtT-20 cells) were doubly labeled by immunofluorescence with specific rabbit antibodies to the alpha-subunits of G proteins (Gs, Gi1-3, or Gq) and monoclonal antibodies against pituitary hormones (GH, PRL, beta LH, beta TSH, or beta FSH) to identify specific cell types. Gs, Gi, and Gq were detected in all secretory types, but differences were found in their levels of expression and cellular distribution. The cell periphery was the predominant site of localization of all these G proteins. In addition, both Gi3 and at least one member of the Gq family were seen by immunofluorescence on intracellular sites typical of the Golgi region. By immunogold labeling, alpha i3 was localized to membranes of Golgi cisternae in sections of rat pituitary and GH3 cells, where it was concentrated on the cis-side of the Golgi stack. These findings demonstrate that G proteins are widely expressed in anterior pituitary cells and further support a role for Gi alpha 3, and possibly Gs, in intracellular membrane trafficking.