The crayfish optic lobe contains high levels of acetylcholine (ACh) and choline as measured with a chemiluminescent assay in small fragments of optic lobe tissue. The highest concentrations were found in the medulla externa and medulla interna (second and third optic neuromeres), which have ACh concentrations of 270 pmol/mg tissue. This concentration is about 16 times that measured in the photoreceptors and lamina ganglionaris (the first optic neuromere). Immunocytochemistry (based upon antisera to choline-glutaryl-BSA) revealed low levels of ACh-like reactivity in the lamina ganglionaris associated with the terminal arbors of centrifugal and/or tangential neurons. The most intense ACh- like reactivity was observed in monopolar neurons of the medulla externa and medulla interna. One monopolar neuron/medullary column (or about 2500 neurons/medullary neuropile) exhibited reactivity and an estimated cytoplasmic concentration of 8.1 mM.