Acetylcholine allows the elicitation of visually evoked behaviors mediated by the frog optic tectum, but the mechanisms behind its effects are unknown. Although nicotinic acetylcholine receptors (nAChRs) exist in the tectum, their subtype has not been assessed. By using quantitative autoradiography, we examined the binding of [(3)H]cytisine and [(125)I]alpha-bungarotoxin in the laminated tectum. In mammalian systems, these radioligands bind with high affinity to alpha4 nAChR subunits and alpha7 nAChR subunits, respectively. [(3)H]Cytisine demonstrated high specific binding in adult frogs in retinorecipient layer 9, intermediate densities in layer 8, and low binding in layers 1-7 of the tectum. [(3)H]Cytisine binding was significantly higher in the tecta of adults than in those of tadpoles. Lesioning the optic nerve for 6 weeks decreased [(3)H]cytisine binding in layers 8/9 by 70+/-1%, whereas 6-month lesions decreased binding by 76+/-3%. Specific binding of [(125)I]alpha-bungarotoxin in adults was present only at intermediate levels in tectal layers 8 and 9, and undetectable in the deeper tectal layers. However, the nucleus isthmi, a midbrain structure reciprocally connected to the tectum, exhibited high levels of binding. There were no significant differences in tectal [(125)I]alpha-bungarotoxin binding between tadpoles and adults. Six-week lesions of the optic nerve decreased tectal [(125)I]alpha-bungarotoxin binding by 33+/-10%, but 6-month lesions had no effect. The pharmacokinetic characteristics of [(3)H]cytisine and [(125)I]alpha-bungarotoxin binding in the frog brain were similar to those demonstrated in several mammalian species. These results indicate that [(3)H]cytisine and [(125)I]alpha-bungarotoxin identify distinct nAChR subtypes in the tectum that likely contain non-alpha7 and alpha7 subunits, respectively. The majority of non-alpha7 receptors are likely associated with retinal ganglion cell terminals, whereas alpha7-containing receptors appear to have a different localization.