Cannon described in 1902 the segmentation motor activity of the small intestine (Canon WB. J Med Res 7: 72–75, 1902). This motor pattern can arise when low-frequency transient depolarizations are evoked in the interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) network, which then affect the omnipresent slow wave activity: changing its regular amplitude into a waxing and waning pattern. The objective of the present study was to investigate physiological stimuli that could induce the low-frequency component. Intracellular recordings were obtained from circular muscle with or without attached mucosa. Decanoic acid (1 mM) and butyric acid (10 mM) both evoked low-frequency transient depolarizations but through different mechanisms. Decanoic acid-induced waxing and waning was initiated by purely myogenic means when perfused onto exposed circular muscle. Butyric acid required the intact mucosa and uninhibited neural activity to elicit the low-frequency response. Evidence is provided that the transient rhythmic depolarizations occur in the absence of interstitial cells of Cajal associated with the myenteric plexus (ICC-MP). Onset of the slow transient depolarizations was stimulated by addition of Nω-nitro-l-arginine (l-NNA; 100 μM); thus the low-frequency component seems to be under chronic inhibition by nitric oxide. Excitatory tachykinergic stimulation induced the low-frequency component since substance P (0.5 μM) evoked it in the presence of neural blockade. In summary, interplay between two networks of myogenic pacemakers, neural activity, and nutrient factors such as fatty acids plays a role in the generation of the rhythmic low-frequency component that is essential for the development of the checkered segmentation motor pattern.