The neuropeptide substance P (SP) stimulates CFU in bone marrow (BM) cultures. Although the methylcellulose matrix used in these assays does not provide an appropriate substratum to support adherent-dependent cells, we have observed that cultures containing optimal SP (10(-8)-10(-10) M) develop confluent areas of reticular/fibroblastoid-like cells with CFUs predominantly localized within their vicinity. Characterization (cytochemical and immunofluorescence) of the reticular/fibroblastoid-like cells indicated that they were fibroblasts, the major constituent of the BM stroma. Hemopoietic effects by SP are mediated by the stroma that expresses SP receptors. We studied the effects of SP (10(-7)-10(-11) M) with suboptimal platelet-derived growth factor-BB (PDGF-BB; 5 ng/ml) and IL-1alpha (2 ng/ml), two fibrogenic cytokines, and also hemopoietic regulators. SP by itself and in synergy with either cytokine induced fibroblast proliferation. At optimum SP, IL-1alpha induced 1.6 times the proliferation of PDGF-BB (87 +/- 7 vs 55 +/- 5; n = 12; p < 0.05). The effects of SP were blunted by a specific neurokinin-1 antagonist. Scatchard analysis indicated that SP binds to BM fibroblasts with an approximate Kd of 5 nM. SP induced steady state mRNA for IL-1 receptor IL-1RI and PDGF-BB (PDGF-AR, PDGF-BR) receptors by 7.5-, 6.2-, and 10.5-fold, respectively. Their up-regulation may be partly responsible for the synergistic effects of SP and their ligands. Induction (3-fold) of neurokinin-1 mRNA by IL-1alpha compared with no induction by PDGF-BB may explain the preferred synergism between SP and IL-1alpha. This study indicates that induction of SP, IL-1alpha, and PDGF-BB receptors is important to their synergistic effects on BM fibroblast proliferation. These results bring new insights into stroma-mediated hemopoietic regulation.