Neuronal survival in vivo may be determined by the combined effects of multiple agents rather than simply by the effect of an individual agent. This idea is supported by experimental evidence showing that neuronal survival can independently be influenced by target-derived factors as well as afferent inputs. To test this idea directly, cultured chick ciliary ganglion neurons were used to study the potentially interactive and combinatorial effects of trophic factors (acidic and basic fibroblast growth factor; FGF), depolarization (as would be expected from afferent activity), and substrate (laminin and collagen IV). Our results were consistent with the idea that combinatorial interactions between multiple agents may be critical in the regulation of cell survival. Exposure to either basic FGF (bFGF) or depolarization on a laminin substrate promoted neuron survival. However, bFGF did not promote survival and depolarization-mediated survival was significantly reduced when neurons were plated on collagen. The simultaneous addition of FGF and depolarization affected survival synergistically when plated on both laminin and collagen. Surprisingly, while survival by FGF or depolarization alone was greatly dependent on substrate, the simultaneous addition of FGF and depolarization appeared to greatly reduce this dependency on substrate. Taken together, these data demonstrate the potential importance of synergistic interactions between trophic factors and depolarizing stimuli.