Blockade of electrical activity in dissociated spinal cord cultures results in a significant loss of neurons during a critical period in development. Decreases in neuronal cell numbers and 125I-labeled tetanus toxin fixation produced by electrical blockade with tetrodotoxin (TTX) were prevented by addition of vasoactive intestinal peptide (VIP) to the nutrient medium. The most effective concentration of VIP was 0.1 nM. At higher concentrations, the survival-enhancing effect of VIP on TTX-treated cultures was attenuated. Addition of the peptide alone had no significant effect on neuronal cell counts or tetanus toxin fixation. With the same experimental conditions, two closely related peptides, PHI-27 (peptide, histidylisoleucine amide) and secretin, were found not to increase the number of neurons in TTX-treated cultures. Interference with VIP action by VIP antiserum resulted in neuronal losses that were not significantly different from those observed after TTX treatment. VIP10-28, a fragment that inhibits VIP stimulation of adenylate cyclase, also produced a dose-dependent decrease in neuronal cell counts similar to that seen with TTX treatment. These data indicate that under conditions of electrical blockade a neurotrophic action of VIP on neuronal survival can be demonstrated.