The GH-releasing hormone (GRH) gene, along with those of many other hypothalamic hormones, is abundantly expressed in mouse and rat placenta. The presence of GRH immunoreactivity (GRH-IR) is described in mouse placenta, maternal blood, and amniotic fluid, and its molecular form has been characterized using HPLC. Two different molecular forms of mouse GRH-IR (mGRH-IR) were detected in the mouse hypothalamus and one in placenta. Twenty-five percent of mGRH-IR in the hypothalamus corresponded to mGRH(1-42)OH, whereas the remainder, and all of the mGRH-IR in placenta, had a retention time consistent with the GRH precursor. High levels of mGRH-IR were detected in both maternal plasma and amniotic fluid. In addition, a mouse placental cell primary culture system was established to study the regulation of mGRH-IR release. Turnover of mGRH in placental cells was rapid, resulting in a 24-h media content of 10 times that present in cells. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol significantly stimulated the release of mGRH-IR from cultured placental cells into the incubation media but had no effect on total peptide synthesis. These results suggest that the release of mGRH-IR from placental cells is mediated, at least in part, by the activation of protein kinase C. The HPLC elution profiles of mGRH-IR released from placental cells under basal and 1-oleoyl-2-acetyl-sn-glycerol-stimulated conditions were similar to those in placental tissue. Although the biological function of mGRH-IR in placental, maternal plasma, and amniotic fluid is not yet clear, the presence of mGRH-IR in these tissues and circulating fluids suggests the possibility that mGRH-IR may exert an important role in both fetal and maternal physiology.