The motor nervous system of adult ascidians consists of neurons forming the cerebral ganglion from which axons run out directly to the effectors, i.e., muscular and ciliary cells. In this study, we analyzed the development of the motor fibers, correlating this with organ differentiation during asexual reproduction in Botryllus schlosseri. We used a staining method for acetylcholinesterase, whose reaction product is visible with both light and electron microscopy and which labels entire nerves, including their thin terminals, making them identifiable between tissues. While the cerebral ganglion is forming, the axons elongate and follow stereotypical pathways to reach the smooth muscle cells of the body, the striated muscle of the heart, and the ciliated cells of the branchial stigmata and the gut. A strict temporal relation links the development of the local neural network with its target organ, which is approached by nerves before the effector cells are fully differentiated. This process occurs for oral and cloacal siphons, branchial basket, gut, and heart. Axons grow through the extracellular matrix and arrive at their targets from different directions. In some cases, the blood sinuses constitute the favorite roads for growing axons, which seem to be guided by a mechanism involving contact guidance or stereotropism. The pattern of innervation undergoes dynamic rearrangements and a marked process of elimination of axons, when the last stages of blastogenesis occur. The final pattern of motor innervation seems to be regulated by axon withdrawal, rather than apoptosis of motor neurons. J. Comp. Neurol. 443:124–135, 2002. © 2002 Wiley-Liss, Inc.