ABSTRACT The Src family of protein tyrosine kinases have been implicated as important regulators of cellular proliferation, differentiation and function. In order to understand further the role of Src family kinases, we have generated loss-of-function mutations in Src64, one of two Src family kinases known in Drosophila melanogaster. Animals with reduced Src64 function develop normally and are fully viable. However, Src64 female flies have reduced fertility, which is associated with the incomplete transfer of cytoplasm from nurse cells to the developing oocyte. Analysis of Src64 egg chambers showed defects in the ring canals that interconnect the oocyte and its 15 associated nurse cells. Src64 ring canals fail to accumulate the high levels of tyrosine phosphorylation that are normally present. Despite the reduced tyrosine phosphorylation, known ring canal components such as filamentous actin, a ring canal-specific product of the hu-li tai shao gene, and the kelch protein localize properly. However, Src64 ring canals are reduced in size and frequently degenerate. These results indicate that Src64 is required for the proper growth and stability of the ovarian ring canals.