The isolated double-track layer of a gram-negative marine pseudomonad was subjected to a number of treatments designed to extract or to digest either phospholipid or protein. Extraction with chloroform–methanol caused aggregation and disruption of the double-track vesicles, a loss of the cleavage plane in frozen preparations, and a disappearance of the ring-like X-ray diffraction pattern which indicates side-by-side packing of the hydrocarbon tails of the phospholipids. Digestion with phospholipase-C, following ethylenediaminetetraacetate (EDTA) treatment, had the same effects except that the diglycerides freed by the action of the enzyme formed paracrystalline lamellar aggregates. This suggests that phospholipids are very important to the structural integrity of the double-track layer, that the layer of phospholipids is centrally located, and that this component of this membrane-like structure is responsible for the X-ray diffraction pattern produced by this material.Protein depletion by urea treatment or by pronase digestion causes increased aggregation of vesicles but these structures are not significantly disrupted, the cleavage plane is present in a stronger form in frozen material, and the X-ray diffraction pattern is unchanged. This suggests that proteins are superficially located and not as important as phospholipids in the structural integrity of this layer.The effects of protein depletion and of phospholipid depletion on the double-track layer are very similar to the effects of the same treatments on membranes, which supports the suggestion that the double-track layer has a membrane-like molecular architecture. The suggestion that this layer constitutes the barrier layer in the gram-negative cell wall is supported by our observations that sodium lauryl sulfate (SLS), EDTA, and polymyxin, which affect the penetrability of the cell wall of whole cells, have been shown to affect the molecular architecture of the isolated double-track layer used in these studies.