Drosophila mutants black and ebony show pigmentation defects in the adult cuticle, which disclose their cooperative activity in β-alanyl-dopamine formation. In visual signal transduction, Ebony conjugates β-alanine to histamine, forming β-alanyl-histamine or carcinine. Mutation of ebony disrupts signal transduction and reveals an electroretinogram (ERG) phenotype. In contrast to the corresponding cuticle phenotype of black and ebony, there is no ERG phenotype observed when black expression is disrupted. This discrepancy calls into question the longstanding assumption of Black and Ebony interaction. The purpose of this study was to investigate the role of Black and Ebony in fly optic lobes. We excluded a presynaptic histamine uptake pathway and confirmed histamine recycling via carcinine formation in glia. β-Alanine supply for this pathway is independent of enzymatic synthesis by Black and β-alanine synthase Pyd3. Two versions of Black are expressed in vivo. Black is a specific aspartate decarboxylase with no activity on glutamate. RNA in situ hybridization and anti-Black antisera localized Black expression in the head. Immunolabeling revealed expression in lamina glia, in large medulla glia, in glia of the ocellar ganglion, and in astrocyte-like glia below the ocellar ganglion. In these glia types, Black expression is strictly accompanied by Ebony expression. Activity, localization, and strict coexpression with Ebony strongly indicate a specific mode of functional interaction that, however, evades ERG detection. J. Comp. Neurol., 521:1207–1224, 2013. © 2012 Wiley Periodicals, Inc.