This investigation examined which signal pathways are of relevance in growth factor-stimulated bone cell mitogenesis. Platelet-derived growth factor (PDGF) and insulin-like growth factor-II (IGF-II) were potent mitogens for both the MG-63 osteoblast cell line and for primary cultures of human osteoblasts (HObs). The mitogenic action of both IGF-II and PDGF was attenuated by pertussis toxin (Ptx), by indomethacin, and by the lipoxygenase inhibitors BW755C74 and BW4AC. A combination of Ptx and indomethacin caused much greater inhibition but failed to abolish mitogenesis completely. PDGF significantly elevated inositol phosphates levels in both cell types; IGF-II had no effect on this pathway. In MG-63 cells, we demonstrated tyrosine phosphorylation of high-molecular-weight substrates elicited by both PDGF and IGF-II. Genistein inhibited the phosphorylation and mitogenic response to PDGF, but had no effect on IGF-II-induced tyrosine phosphorylation or mitogenesis. Another inhibitor of tyrosine kinases, methyl 2,5-dihydroxycinnamate, (MDHC), inhibited PDGF-stimulated mitogenesis effectively in both cell types but only blocked IGF-II-induced mitogenesis in MG-63 cells. The specificity of these inhibitors suggests that particular tyrosine kinases may regulate growth factor-induced stimulation of bone cells.