In Drosophila melanogaster, nanos functions as a localized determinant of posterior pattern. Nanos RNA is localized to the posterior pole of the maturing egg cell and encodes a protein that emanates from this localized source. Nanos acts as a translational repressor and thereby establishes a gradient of the morphogen Hunchback. Here we show that the mechanism by which nanos acts in Drosophila is a common developmental strategy in Dipteran insects. We used cytoplasmic transplantation assays to demonstrate that nanos activity is found in posterior poleplasm of five diverse Dipteran species. Genes homologous to nanos were identified from Drosophila virilis, the housefly Musca domestica, and the midge Chironomus samoensis. These genes encode RNAs that are each localized, like nanos, to the embryonic posterior pole. Most importantly, we demonstrate that these homologues can functionally substitute for nanos in D. melanogaster. These results suggest that nanos acts in a similar pathway for axis determination in other insects. Comparison of the Nanos sequences reveals only 19% overall protein sequence similarity; high conservation of a novel zinc finger near the carboxy terminus of the protein defines a region critical for nanos gene function.