The electrophysiological properties of guinea pig peritoneal macrophages cultured in vitro were studied using standard intracellular recording techniques. The mean transmembrane potential, input resistance and time constant recorded from these cells were -13.1 mV, 140 Mohms, and 18 msec respectively. The majority of macrophages exhibited spontaneous hyperpolarizations (HA) of 4-8 seconds in duration and 10-50 mV in amplitude. Mouse peritoneal macrophages and human monocyte-derived macrophages manifested similar HA. HA could be induced by either mechanical stimulation or application of hyperpolarizing currents of 2-8 namps. HA had a mean reversal potential of -53 mV. Increasing the extracellular [ K + ] 10-fold resulted in a 50 mV shift in reversal potential. Addition of EGTA (1.5 mM) inhibited both spontaneous and evoked macrophage HA in the presence of excess Mg+ +. The divalent cation ionophore, A23187 induced prolongation of HA at low concentration (0.6 X and resulted in sustained hyperpolarization at higher concentration (2.0 X M ) . Addition of EGTA to cells treated with A23187 abolished HA. These data indicate that: ( 1 ) cultured macrophages from a variety of species exhibit spontaneous and induced HA, ( 2 ) development of HA is related to an increase in membrane permeability to K f , and ( 3 ) C a + + may regulate the spontaneous and evoked electrical activity of the macrophage membrane presumably by affecting K + permeability, M )