Gastrin-releasing polypeptide (GRP) has been implicated in the development of the human fetal lung. To determine whether GRP has a wider role in fetal development, its actions on DNA synthesis and cell replication by isolated epiphyseal growth plate chondrocytes obtained from ovine fetuses between 35 days gestation and near term (145 days) were examined. Chondrocytes were isolated using collagenase from the proximal tibia and cultured in monolayer. Synthesis of DNA was assessed from the incorporation of [3H]thymidine into previously growth-restricted cells after incubation in medium supplemented with GRP1-27 (40-1280 nM). Increase in cell number was assessed after incubation with test medium for 1 week. GRP caused a dose-dependent increase in both cell number and DNA synthetic rate compared to control incubations. Cell number was increased by 50% in the presence of a maximally effective 160 nM GRP and DNA synthesis by up to 800% utilizing chondrocytes obtained from animals of 75-80 days gestation. The mean (+/- SEM) half-maximal concentration of GRP for the stimulation of DNA synthesis was 97 +/- 12 nM (5 separate fetuses). Concentrations of GRP in excess of 160 nM caused a sharp reduction in both cell replication and DNA synthesis. To determine where within the cell cycle GRP exerted its mitogenic action, synchronized chondrocytes were transiently exposed to fetal bovine serum and cultured with GRP for increasing periods of time before pulse labeling with [3H]thymidine during S phase. GRP was as effective in stimulating DNA synthesis when present for the initial 4 h of G1 as when present for the entire G1 period. Since isolated fetal growth plate chondrocytes release insulin-like growth factor II (IGF II) and basic fibroblast growth factor (basic FGF) the possible mediation of GRP action by the release of these peptides or synergistic interactions were examined. Specific antibodies shown to negate the mitogenic actions of exogenous IGFs or basic FGF on chondrocytes did not alter GRP-stimulated DNA synthesis. The release of radioimmunoassayable IGF II by chondrocytes was not altered in the presence of GRP. Coincubation of GRP with submaximal concentrations of IGF I or basic FGF showed additive effects on DNA synthesis. When the actions of galanin were examined it was found to inhibit basal DNA synthesis by chondrocytes at a concentration of 167 nM. However, 66 nM or greater galanin was able to render 160 nM GRP inactive as a mitogen. These results suggest that GRP may potentially influence skeletal development in the ovine fetus and may interact with locally released peptide growth factors or other neuropeptides.