At embryonic stage 16, Myo61F protein is concentrated at the basolateral domain of immature enterocytes and is diffusely located in the cytoplasm. At embryonic stage 17, intense apical staining is observed in most regions of the gut. In larvae and adults, Myo61F protein is found in the brush border predominantly in the apical microvillar domain. In egg chambers, it is found in the basolateral domain of follicle cells until stage 8 after which it is more apically positioned. In stage 10, the localization of Myo61F protein to the api al domain of columnar follicle cells near the oocyte is thought to correspond to the microvilli of the brush border which exists there.

Myo61F protein is localized to the brush border of enterocytes through the entire length of the larval midgut. It is also localized to the brush borders of specialized secretory cells of the digestive sytem, including the proventriculus, gastric ceaca, cuprophilic cells, and in the apical domain of epithelial cells of Malphigian tubules. In adults, Myo61F is expressed in high levels in the inner chiasm of the optic lobe, with significant expression in the lamina and medulla. Myo61F is also expressed in photoreceptors, where it is concentrated at the base of microvilli.

Myo61F protein is first detected on western blots in 12-16hr embryos, increases dramatically at the end of embryogenesis and in first instar larvae, increases slightly in later larval stages, is present at low levels in pupae and is observed at higher levels in adults. Myo61F protein is detected by immunolocalization in the alimentary canal and its derivatives and in egg chambers. In stage 17 embryos, it is observed in the gastric caeca but not in the foregut. A gradient of expression is observed in the midgut with most of the staining in the posterior midgut and increasingly less in the middle midgut and anterior midgut. Some protein is present in the hindgut. In egg chambers, protein is present in follicle cells. By stage 10, it is associated only with columnar follicle cells surrounding the oocyte. Additionally, it is associated with centripetally migrating follicle cells over the anterior of the oocyte.