Mouse spinal neurons grown in tissue culture were used to examine the membrane mechanisms of action of the peptide substance P. Two functionally distinct actions were observed, one being a rapidly desensitizing excitation, and the other being a dose-dependent, reversible depression of excitatory responses to the putative amino acid neurotransmitter glutamate. These effects on excitability suggest that substance P may play more than one role in intercellular communication in the nervous system.