Effects of divalent cations on oscillations of membrane potentials (i.e., spontaneous repetitive hyperpolarizing responses) and on hyperpolarizing responses induced by electrical stimuli as well as on resting potentials were studied in large nondividing L cells. Deprivation of Ca2+ from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential Sr2+ or Mn2+ applied to the external medium in place of Ca2+ was able to substitute for Ca2+ in the generation of hyperpolarizing responses, while Mg2+, Ba2+ or La3+ suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca2+, Sr2+, Mn2+ or La3+ induced membrane hyperpolarization. When the external Ca2+, Sr2+ or Mn2+ concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca2+ was of the same order of magnitude as those of hyperpolarizing responses and was dependent on the external K+ concentration. In the light of these experimental observations, it was deduced that the K+ conductance increase associated with the hyperpolarizing excitation is the result of an increase in the intracellular concentration of free Ca2+ mainly derived from the external solution.