The first expression and distribution of acetylcholinesterase (AChE) activity was studied among a distinct population of early neurons in embryonic zebrafish by using histochemical and retrograde labeling techniques. AChE first appeared in the nervous system in the primary motoneurons of the rostral spinal cord when the embryo had nine somites, approximately 14 hours postfertilization. Subsequent expression of AChE activity in the spinal cord proceeded in a rostral-to-caudal sequence. Cranial neurons expressed AChE activity shortly after it appeared in the rostral spinal cord. Several hours later, near the end of the first day, primary neurons in the hindbrain and spinal cord all contained AChE, including sensory neurons, reticulospinal interneurons, and primary motoneurons. AChE activity was also detected in the nucleus of the medial longitudinal fasciculus. Presumptive cranial ganglia transiently expressed AChE activity between 14 and 24 hours of development. These results, combined with previous observations that examined the time of origin and axogenesis of primary neurons, suggest that primary neurons in the embryonic zebrafish contain AChE before they sprout axons. The primary neurons appear to follow a common sequence of development consisting of a withdrawal from the cell division cycle, the expression of AChE, and axogenesis. Although this sequence is followed by all primary neurons, lack of a rostral-to-caudal sequence in the time of birth and variability in the time of axon outgrowth demonstrate that the relative timing of these three events is not rigidly programmed in individual neurons. Moreover, the very early expression of AChE in such diverse cell types suggests that it may have a developmental role in addition to its function in transmitter metabolism.