To gain insight into the transmissibility of bacteria by house flies, the temporal and spatial fate of green fluorescent protein (GFP)-expressing motile and nonmotile strains of Aeromonas hydrophila was examined within the alimentary canal. Liquid food consumed by house flies is first stored in the crop and then is regurgitated and/or passed into the midgut. Once within the midgut, food is contained inside a double-layered peritrophic matrix (PM), with the inner layer enveloping digested material and forming fecal pellets for excretion. Between 1 and 12 h after ingestion, and irrespective of motility, live GFP A. hydrophila adhered to the luminal surfaces of the crop and inner PM of bacteria-fed flies. However, some nonadherent, motile bacteria moved freely within the PM lumen in the anteromedial midgut, whereas fecal pellets (lysed bacteria) continued passing posteriorly. At 12–24 h, adhered bacteria of both strains were lysed in the distal midgut, compressed into fecal pellets, and excreted. Viable bacteria in the crop visually exceeded numbers within these fecal pellets. Culture recovery at the same time points showed a 1,000-fold increase of viable bacteria at 2 h, presumably in the crop, with a temporal decline thereafter. Further, viable bacteria were recovered from vomit specks and orally contaminated substrates up to 2 h after feeding but never from feces. These results suggest that A. hydrophila is a transient resident of the house fly alimentary canal and is only orally transmissible for a short time after ingestion. Thus, regurgitation may be more significant than fecal transmission in the spread of some house fly transmitted bacterial diseases.