Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.