SummaryThree different techniques, protease accessibility, ceil fractionation andin situimmunocytochemistry, were used to study the location of the lipoprotein pullulanase produced byEscherichia coliK12 carrying the cloned pullulanase structural gene (pulA)fromKlebsiella pneumoniae, with or without theK. pneumoniaegenes required to transport pullulanase to the cell surface (secretion‐competent and secretion‐incompetent, respectively). Pullulanase produced by secretion‐competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked theN‐terminal fatty‐acylated cystelne residue (and probably also a shortN‐terminal segment of the pullulanase polypeptide), confirming that theN‐terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion‐incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electron‐microscopy of vesicles labelled with antipullulanase serum and protein A‐gold confirmed that pullulanase was associated with both types of vesicles. When thin‐sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion‐competent cells and mainly in the proximity of the inner membrane in the secretion‐incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility andin situimmunocytochemistry) show that pullulanase is transported to the cell surface of secretion‐competent cells, this could not be confirmed by cell‐fractionation techniques. Possible explanations for t