Color discrimination requires the input of different photoreceptor cells that are sensitive to different wavelengths of light. TheDrosophila visual system contains multiple classes of photoreceptor cells that differ in anatomical location, synaptic connections, and spectral sensitivity. The Rh5 and Rh6 opsins are expressed in nonoverlapping sets of R8 cells and are the onlyDrosophila visual pigments that remain uncharacterized. In this study, we ectopically expressed Rh5 and Rh6 in the major class of photoreceptor cells (R1–R6) and show them to be biologically active in their new environment. The expression of either Rh5 or Rh6 in “blind” ninaE17 mutant flies, which lack the gene encoding the visual pigment of the R1–R6 cells, fully rescues the light response. Electrophysiological analysis showed that the maximal spectral sensitivity of the R1–R6 cells is shifted to 437 or 508 nm when Rh5 or Rh6, respectively, is expressed in these cells. These spectral sensitivities are in excellent agreement with intracellular recordings of the R8p and R8y cells measured inCalliphora and Musca. Spectrophotometric analyses of Rh5 and Rh6 in vivo by microspectrophotometry, and of detergent-extracted pigments in vitro, showed that Rh5 is reversibly photoconverted to a stable metarhodopsin (λmax = 494 nm), whereas Rh6 appears to be photoconverted to a metarhodopsin (λmax = 468 nm) that is less thermally stable. Phylogenetically, Rh5 belongs to a group of short-wavelength-absorbing invertebrate visual pigments, whereas Rh6 is related to a group of long-wavelength-absorbing pigments and is the first member of this class to be functionally characterized.