Intracellular recording methods were used to investigate the ionic basis for the postsynaptic actions of substance P (SP) on mouse spinal cord neurons grown in primary dissociated cell culture. SP and an analog, eledoisin-related peptide (ERP), were applied to single neurons by pressure ejection of peptide-containing solutions from blunt (2- to 10-micrometers) glass micropipettes. SP and ERP had similar excitatory actions, increasing spontaneous activity and depolarizing neurons by decreasing membrane conductance. Depolarizing responses were not inverted by intracellular injection of chloride ions, suggesting that SP responses did not result from decreased chloride conductance. SP and ERP responses were not abolished by extracellular tetraethylammonium ions (TEA+) but were reduced or eliminated by intracellular TEA+, suggesting that SP reduced a potassium conductance (gK). Finally, SP and ERP responses were larger when neurons were depolarized and smaller when the cells were hyperpolarized, and extrapolated reversal potentials for the peptide responses were 10 to 30 mV more negative than resting membrane potential. Thus, it was concluded that SP depolarized spinal cord neurons by decreasing a membrane potassium conductance. However, SP and ERP response polarity was not clearly reversed even at membrane potentials more negative than the expected potassium equilibrium potential. Moreover, extrapolated reversal potentials (RPeS) of SP responses varied linearly with the logarithm of extracellular potassium concentration ([K+]o) as predicted by the Nernst equation for potassium in [K+]o concentrations of 10, 15, 20, and 40 mM but were more depolarized than predicted for [K+]o concentrations of 1 and 5 mM. Since reduction of extracellular sodium by choline substitution did not alter the deviation from the Nernst equation for potassium, it was concluded that SP decreased a voltage- dependent potassium conductance which was absent at very negative potentials, present at resting membrane potential, and activated by membrane depolarization. Thus, SP decreases a conductance which appears similar to the muscarine-sensitive potassium conductance in sympathetic ganglion neurons (Brown, D. A., and P. R. Adams (1980) Nature 283: 673–676).