In a previous paper (A. Verkleij, L. van Alphen, J. Bijvelt, and B. Lugtenberg, Biochim. Biophys. Acta 466:269-282, 1977) we have hypothesized that particles on the outer fracture face of the outer membrane ([Formula: see text]), with corresponding pits on the inner fracture face of the outer membrane ([Formula: see text]), consist of lipopolysaccharide (LPS) aggregates stabilized by divalent cations and that they might contain protein and/or phospholipid. In the present paper the roles of LPS, cations, and proteins in these [Formula: see text] particles are described more extensively, using a strain that lacks the major outer membrane proteins, b, c, and d (b− c− d−), and has a reduction in the number of [Formula: see text] particles of 75%. To study the role of divalent cations in the formation of [Formula: see text] particles, these b− c− d− cells were grown or incubated with Ca2+, Mg2+, or putrescine. The presence of Ca2+ resulted in the appearance of many [Formula: see text] particles and [Formula: see text] pits. Mg2+ and putrescine were less effective than Ca2+. Introduction of these particles was not accompanied by alterations in the relative amounts of LPS and cell envelope proteins. Ca2+ treatment of a heptoseless derivative of a b− c− d− strain did not result in morphological changes. Incubation of Ca2+-treated cells with ethylenediaminetetraacetate caused the disappearance of the introduced particles as well as the release of more than 60% of the cellular LPS. These results strongly support the hypothesis that LPS is involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The roles of various outer membrane proteins in the formation of [Formula: see text] particles were studied by comparing the freeze-fracture morphology of b− c− d− cells with that of cells which contain one of the outer membrane proteins b, c, d, and e or the receptor protein for bacteriophage lambda. The results showed that the presence of any of these five proteins in a b− c− d− background resulted in a large increase in the number of [Formula: see text] particles and [Formula: see text] pits, indicating that these proteins are, independent of each other, involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The simplest explanation for the results is that in wild-type cells each particle consists of LPS complexed with some molecules of a single protein species, stabilized by either divalent cations or polyamines. It is hypothesized that the outer membrane of the wild-type cell contains a heterogeneous population of particles, of which 75% consists of protein b-LPS, protein c-LPS, and protein d-LPS particles. A function of these particles as aqueous pores is proposed.