Copper cells were originally identified in Drosophila midgut epithelium by their striking orange fluorescence in copper-fed larvae. Here, we examined copper cell fluorescence in light of the previous observations that (1) a similar fluorescent signal in yeast is produced by a complex between copper and metallothionein, and (2) metallothionein is expressed constitutively in the copper cell region and inducibly in other regions of the Drosophila midgut. Pulse-feeding experiments with 1 mM CuCl2 revealed that fluorescence appeared rapidly in copper cells (<5 min) and slowly in other cells of the midgut (days), suggesting a constitutive cofactor in the former and an inducible cofactor in the latter. Fluorescence was also detected in Drosophila S2 tissue culture cells after induction of metallothionein synthesis by addition of CuCl2 to the growth medium. Thus, fluorescence coincided spatially and temporally with the expression of metallothionein. Fluorescence was also linked to the acid-secreting activity of copper cells. Fluorescence was not observed when acid secretion was inhibited by a mutation in the alpha spectrin gene and acidification was blocked in copper-fed wild-type larvae. However, acidification was restored after a 1-day chase period in which the fluorescent signal became sequestered within a vesicular compartment. We therefore conclude that copper cell fluorescence is most probably attributable to a cytoplasmic copper-metallothionein complex, suggesting an unanticipated role for metallothionein in acid-secreting cells.