We have previously demonstrated that neuropeptide Y (NPY) inhibits voltage sensitive Ca2+ channels in rat dorsal root ganglion neurons and that this effect is mediated by a pertussis toxin-sensitive, guanyl nucleotide-binding protein (G-protein). We now demonstrate that NPY can also stimulate the synthesis of inositol trisphosphate (InsP3) and diacylglycerol in dorsal root ganglion neurons. The effects of NPY were compared with those of bradykinin (BK) which also stimulates phosphoinositide turnover in these cells. NPY-stimulated InsP3 synthesis could be completely blocked by treatment with pertussis toxin and significantly enhanced by cholera toxin although not by other agents which raised cellular concentrations of cyclic AMP. In contrast, the effects of BK were completely unaltered by either toxin. Furthermore the maximal effects of BK and NPY were additive. In spite of the lack of toxin effects, stimulation of InsP3 synthesis produced by BK was clearly mediated by a G-protein. Thus BK stimulated InsP3 production in digitonin-permeabilized neurons, and these effects were enhanced by guanosine 5'-O-(3-thiotriphosphate) and blocked by guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of both NPY and BK were also blocked by treatment of neurons with phorbol esters. Fura-2-based microfluorimetry of single dorsal root ganglion neurons demonstrated that both BK and NPY increased cytoplasmic-free Ca2+ concentration and that both peptides could produce this effect in the same neuron. Both agents could still increase cytoplasmic-free Ca2+ concentration in Ca2+-free medium indicating that the source of the Ca2+ was an intracellular store. Thus, both NPY and BK can activate InsP3 synthesis in the same cell but seem to utilize different G-proteins. NPY utilizes a pertussis toxin-sensitive G-protein and BK a toxin-insensitive one.