The cholinergic system in the brain has been widely studied in most vertebrate groups, but there is no information available about this neurotransmission system in the brains of holostean fishes, a primitive and poorly understood group of actinopterygian fishes. The present study provides the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two holostean species, the Florida gar, Lepisosteus platyrhincus, and the bowfin, Amia calva. Immmunohistochemistry against the enzyme choline acetyltransferase (ChAT) revealed distinct groups of ChAT-immunoreactive (ChAT-ir) cells in the habenula, isthmic nucleus, laterodorsal tegmental nucleus, octavolateral area, reticular formation, cranial nerve motor nuclei and the motor column of the spinal cord, all of which seem to be highly conserved among vertebrates. Some ChAT-ir cells were detected in the basal telencephalon that appear in actinopterygians for the first time in the evolution of this neurotransmission system, whereas the remarkable cholinergic population in the optic tectum is a peculiar characteristic, the presence of which varies throughout evolution, although it is present in all teleosts studied. Abundant cholinergic fibers were found in the pretectal region and optic tectum, where they probably modulate vision, and in the hypothalamus and the interpeduncular neuropil. Some interspecific differences were also observed, such as the presence of ChAT-ir cells in the supraoptoparaventricular band only in Lepisosteus and in in the nucleus subglomerulosus only in Amia. In addition, ChAT-ir fibers in the olfactory bulb were detected only in Amia. Comparison of these results with those from other classes of vertebrates, and a segmental analysis to correlate cell populations, reveal that the pattern of the cholinergic system in holosteans is very close to that in ancestral actinopterygian fishes, as recently described in the bichir (Cladistia), although an important evolutionary novelty in holosteans is the presence of cholinergic cells in the basal telencephalon.