The cytoarchitecture of nuclei in the diencephalon and the distribution of acetylcholinesterase (AChE) in the diencephalon and optic tectum were analyzed in the longnose gar, Lepisosteus osseus, a non-teleost actinopterygian fish. Nuclei were identified in the preoptic area, thalamus, posterior tubercle, hypothalamus, synencephalon, and pretectum which are homologous to like-named nuclei in teleosts and other non-teleost actinopterygians. Of particular note, a nucleus in the rostral diencephalon, nucleus rostrolateralis, which has previously been identified only in the osteoglossomorph Pantodon, is present in the longnose gar. The posterior pretectal nucleus, previously identified in teleosts and in the bowfin Amia, is also present in gars. The small size of the posterior pretectal nucleus in gars supports the hypothesis that this nucleus was small plesiomorphically. The distribution of AChE in the diencephalon and optic tectum corresponds in most respects to that found in teleosts. The superficial pretectal nuclei, including the posterior pretectal nucleus, are strongly positive for AChE. In contrast, most of the nuclei within the preglomerular complex are negative for AChE. Acetylcholinesterase is present in some of the fibers in the optic tracts and in most retinorecipient nuclei, as well as in some other nuclei and tracts.