The MotA protein of Escherichia coli is a component of the flagella that functions together with the MotB protein in transmembrane proton conduction. It is an integral membrane protein, with four hydrophobic segments that might traverse the membrane and two short segments that are predicted to be in the periplasm. In a previous study of the accessibility of MotA to various proteases, evidence for periplasmic segments was not obtained, probably because they are small. Here, we report site-directed sulfhydryl labeling experiments which show that two segments of MotA are exposed on the periplasmic side of the membrane, while the rest of the protein is in the cytoplasm. These experiments establish that the main features of the suggested model for MotA topology are correct, furnishing a basis for more detailed structure-function studies of the MotA/MotB proton channel.