AbstractThe photosensitive microvilli of Drosophila photoreceptors R1–R6 are not aligned in parallel over the entire length of the visual cells. In the distal half of each cell, the microvilli are slightly tilted toward one side and, in the proximal half, extremely toward the opposite side. This phenomenon, termed rhabdomere twisting, has been known for several decades, but the developmental and cell biological basis of rhabdomere twisting has not been studied so far. We show that rhabdomere twisting is also manifested as molecular polarization of the visual cell, because phosphotyrosine‐containing proteins are selectively partitioned to different sides of the rhabdomere stalk in the distal and proximal sections of each R1–R6 photoreceptor. Both the asymmetrical segregation of phosphotyrosine proteins and the tilting of the microvilli occur shortly before eclosion of the flies, when eye development in all other aspects is considered to be essentially complete. Establishment of rhabdomere twisting occurs in a light‐independent manner, because phosphotyrosine staining is unchanged in dark‐reared wild‐type flies and in mutants with defects in the phototransduction cascade, ninaE17 and norpAP24. We conclude that antiphosphotyrosine immunofluorescence can be used as a light microscopic probe for the analysis of rhabdomere twisting and that microvilli tilting represents a type of planar cell polarity that is established by an active process in the last phase of photoreceptor morphogenesis, just prior to eclosion of the flies. J. Comp. Neurol. 498:68–79, 2006. © 2006 Wiley‐Liss, Inc.