Electrical rhythmicity in the gastrointestinal (GI) muscles is generated by pacemaker cells, known as interstitial cells of Cajal (ICC). In the present study, we investigated the effect of external divalent cations on pacemaking activity in cultured ICC from murine small intestine by using whole-cell patch clamp techniques. ICC generated pacemaker currents under a voltage clamp or electrical pacemaker potentials under a current clamp, and showed a mean amplitude of ⁣500⁑50 pA or 30⁑1 mV and the frequency of 18⁑2 cycles/min. Treatments of the cells with external 0 mM Ca2⁢ stopped pacemaking activity of ICC. In the presence of 2 mM Ca2⁢, 0 mM external Mg2⁢ depolarized the resting membrane potential, and there was no change in the frequency of pacemaking activity. However, 10 mM external Mg2⁢ decreased the frequency of pacemaking activity (6.75⁑1 cycles/min, n=5). We replaced external 2 mM Ca2⁢ with equimolar Ba2⁢, Mn2⁢ and Sr2⁢, and they all developed inward current in the sequence of Ba2⁢＞Mn2⁢＞Sr2⁢. Also the frequency of the pacemaking activity was stopped or irregulated. We investigated the effect of 10 mM Ba2⁢, Mn2⁢ and Sr2⁢ on pacemaking activity of ICC in the presence of external 0 mM Mg2⁢, and found that 10 mM Ba2⁢ and Mn2⁢ induced large inward current and stopped the pacemaking activity of ICC (n=5). Interestingly, 10 mM Sr2⁢ induced small inward current and potentiated the amplitude of pacemaking activity of ICC (n=5). These results indicate that extracellular Ca2⁢ and Mg2⁢ are requisite for the pacemaking activity of ICC.