A descriptive enzyme histochemical study on the expression of acetylcholinesterase (AChE) in the developing rat spinal cord is presented. Between E11-E16, AChE was found to be associated with premitotic neurons of the ventral matrix layer, which indicated an involvement in the proliferation of the spinal cord motor neurons. From E12 on, AChE was abundantly present in the motor neurons of the ventral mantle layer, and in their fibres. This early expression suggested a function of AChE in the development of the motor neurons, rather than an active role in cholinergic transmission. The intermediolateral, the intermediomedial cell column and the region between these cell groups, were found to be positive for AChE. Cells of the adult intermediolateral cell column also expressed AChE. AChE, therefore, apparently plays a role in the development as well as in the functioning of the rat autonomic system. In the lateral funiculus, cells of the lateral spinal nucleus expressed AChE. After P8, AChE was expressed in the substantia gelatinosa. The enzyme may be associated with the fibre terminals of the primary afferents. AChE was found to be temporary expressed in the developing dorsal funiculus, which suggested a function of the enzyme in fibre growth and path-finding. At E12, AChE was located in the ventral aspect of the dorsal root ganglion. Later on, AChE positive cells were found throughout the ganglion.