Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.