Piliated Burkholderia (formerly Pseudomonas) cepacia from sputa of cys tic fibrosis patients in Toronto, Canada, were shown earlier to bind to purified mucins and to a protein receptor on epithelial cells via a 22-kDa adhesin located on unique cable pili. However, a second receptor, thought to be lipid in nature, was also identified on cells and appeared to serve as the major cell receptor for poorly piliated or nonpiliated isolates. In the present study in vitro approaches were used to identify putative lipid receptors for B. cepacia and to explore the nature of the binding interaction. As judged by thin-layer chromatography overlay assays, the best receptors were digalactosylceramide and globotriosylceramide (Gb(3)). Both contain and unsubstituted terminal Gal alpha 1-4Gal sequence. B cepacia also bound moderately to galactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking adhesin for GB(3). Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the carbohydrate epitope to the bacteria. Gb(3) was also the major receptor in lipid extracts of human erythrocytes, human buccal epithelial cells and HEp-2 laryngeal epithelial cells. In a receptor-based enzyme-linked immunosorbent assay, binding to Gb(3) within a phospholipid-cholesterol mixture (a membrane-like environment) increased and then approached saturation as a direct function of increasing bacterial concentration. The calculated value of K(a) (3.06 X 10(-8) ml/CFU), the affinity constant, was almost identical to the K(a) calculated earlier for B. cepacia binding to a set of lipid receptors in buccal epithelial cells (1.5 X 10(-8) to 2.0 X 10(-8) ml/CFU). Our findings suggest that within cell membranes, galactose-containing glycolipids, particularly Gb(3) are good candidates for receptors for B. cepacia, particularly for isolates in which cable pili are poorly expressed.