Intestinal motor patterns are not well developed in premature infants. Similarly, in neonatal mice, irregular motor patterns were observed. Pacemaker cells, identified in the small intestine as interstitial cells of Cajal (ICCs) associated with Auerbach's plexus (ICC-APs), contribute to the generation of peristaltic movements. The objective of the present study was to assess the hypothesis that abnormal gut motor activity in (preterm) newborns can be associated with underdeveloped ICCs. Specifically, the aim was to identify at which point the electrical pacemaker activity is fully developed and whether or not the development of pacemaker activity has a structural correlation with the developmental stage of ICCs. Pacemaker activity was identified as that component of the slow wave that is insensitive to L-type calcium (Ca 2Ο© ) channel blockers and displays a characteristic reduction in frequency in the presence of cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum Ca 2Ο© pump. In newborn, unfed neonates, action potentials occurred that were irregular in frequency and amplitude and sensitive to verapamil. CPA (5 Β΅M) abolished all action potentials. Quiescent spots were observed in approximately 50% of impalements. Six hours after birth, slow-wave activity appeared at a regular frequency and amplitude, and a welldefined plateau phase was observed. Verapamil did not affect the frequency, 5 Β΅M CPA decreased it. The effect of CPA on the pacemaker frequency 2 days after birth was identical to that observed in adult mice. In 2-hr-old neonates, ICCs could be identified through selective uptake of methylene blue, but ultrastructural features were not fully developed. At 48 hr, a complete ICC network covering Auerbach's plexus was formed, confirmed by electron microscopy. In summary, the pacemaker component of the slow waves can be identified in neonates as early as 6 hr after birth. The pacemaker component was fully developed 2 days after birth. These electrophysiological observations correlated with the development of full network characteristics of ICC-APs and the development of fully differentiated ICC-APs from ''blast-like'' cells. Dev.