Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca(2+) imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca(2+) transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca(2+) transient wave fronts propagated through the ICC-MY networks at approximately 2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca(2+) transients in LM but had no visible effect on the transients in ICC-MY. beta-Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca(2+) entry (Ni(2+) and mibefradil) and elevated external K(+) reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca(2+) transients in ICC-MY. These data show that global Ca(2+) transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca(2+) handling mechanisms and entrained by voltage-dependent Ca(2+) entry and coupling of cells via gap junctions.