We have analyzed the distribution pattern of choline acetyltransferase (ChAT) in the zebrafish brain and retina during ontogeny. ChAT-immunoreactive (ChAT-ir) neurons are observed in the prosencephalon from 60 h postfertilization (hpf) onwards, exclusively in the preoptic area (basal plate of p6) derived from the secondary prosencephalon. In the mesencephalon, ChAT-ir cells are observed in both the optic tectum and the tegmentum. Stained cells in the tegmentum are observed from 60 hpf onwards, while in the optic tectum they appear after hatching. In the rhombencephalon, ChAT-ir cells are first observed in the isthmic region (rh1) and in the medulla oblongata (rh5-rh7) at the end of embryonic life. The rhombencephalic cholinergic cell groups develop in a gradual caudorostral sequence. Motoneurons of the spinal cord are ChAT-ir from 48 hpf onwards. The retina displays ChAT-ir neuropil in both the inner and outer plexiform layers from embryonic life, whereas stained amacrine cells are only observed after hatching. The staining in the outer plexiform layer gradually decreases during juvenile development. The optic nerve axons show a transient expression of ChAT at the end of embryonic development. The early presence of ChAT immunolabeling suggests an important neuromodulator role for acetylcholine in the first developmental stages.