The in vivo orientation of the channel forming porin OmpF from the outer membrane of Escherichia coli was assessed by immunological, biochemical and structural techniques. Porin OmpF exists as a trimer of channels formed by 16 antiparallel beta-strands. These are connected by long hydrophilic loops on one side of the bilayer and short loops or beta-turns on the other. The former constitute the rough side of the porin channel, the latter the smooth side. Epitopes at the cell surface have all been mapped within the long loops, suggesting a rough-side-out orientation of OmpF in the membrane. We analyzed detergent solubilized OmpF trimers, reconstituted 2-D OmpF crystals, OmpF containing outer membranes (sacculi) and intact cells of an E. coli strain overexpressing OmpF. Both solubilized OmpF and OmpF containing sacculi were exposed to proteases, and distinct cleavage sites were identified by protein sequencing. Solubilized OmpF, reconstituted 2-D OmpF crystals and detergent extracted sacculi were tested for their capacity to adsorb colicin N. We used antibodies directed against surface exposed epitopes for immunogold labeling of reconstituted 2-D OmpF crystals and sacculi. The surfaces of intact cells and extracted sacculi were analyzed by electron microscopy and image processing. Finally, a full 3-D reconstruction of negatively stained OmpF containing sacculi revealed the OmpF trimer in its native conformation within the outer membrane. Colicin N and antibody experiments, as well as the 3-D map of the sacculi demonstrated that OmpF exposes the long loops to the extracellular space. In contrast, reconstituted crystalline OmpF vesicles and double layered sheets were found to be in an inside-out conformation, hence hiding colicin or antibody binding epitopes. Two proteinase K cleavage sites were identified, one on a protruding loop and the other inside the channel on the loop penetrating the pore.