To investigate spontaneous rhythmicity in smooth muscle tissue, we have developed a cell cluster preparation. Cell clusters were enzymatically isolated from the muscle layer of mouse small intestine and cultured for several days. They included smooth muscle, neurones, and c-Kit-immunopositive interstitial cells. c-Kit-immunopositive cells in myenteric plexus, showing a networklike structure, are putative pacemaker cells. The cultured cell clusters routinely show spontaneous contraction and preserve characteristic features in this tissue: (1) high temperature dependency of contractile frequency; (2) spontaneous electrical activities measured with patch clamp techniques are insensitive to tetradotoxin (TTX) and dihydropyridine Ca(2+) antagonists. This preparation could therefore be used as a good model system to investigate the underlying mechanisms of intestinal motility and pacemaker function. The relationship between the frequency of electrical activity and cluster size suggests that the minimum unit of small intestine tissue to yield normal pacemaker activity is approximately 100 microm in diameter, or less. The applications of 100-120 microM Cd(2+) and Ni(2+) significantly suppressed the spontaneous activity. Ca(2+) influx pathways other than L-type and "classical" T-type voltage-sensitive Ca(2+) channels seem very likely to play an important role, such as nonselective cation channels and capacitative Ca(2+) entry. Furthermore, applications of heptanol reduced the amplitude and the frequency of the oscillating inward currents and eventually terminated them, suggesting that electrical cell-to-cell coupling may also make some contribution to the generation of spontaneous activity.