Bacterial adherence to tissues is a necessary step in infection and results from interactions between surface molecules on the bacteria (adhesins) and plasma membrane receptors on host cells. Using a clinical isolate of Pseudomonas aeruginosa, we have examined the receptors required for adherence to Chinese hamster ovary (CHO) cells. In mutant CHO cell lines and mucin-producing lung epithelial cell lines, adherence did not depend on glycoproteins, glycolipids, or proteoglycans. Treating cells with glutaraldehyde and proteases also did not affect adherence. However, treating cells with ethanol diminished adherence and resulted in removal of factors that would facilitate binding of bacteria when coated on plastic culture plates. These factors were purified by solvent extraction and silica gel chromatography and were identified as cholesterol and cholesterol esters by coelution with authentic standards, gas chromatography, 1H NMR, and mass spectrometry. Bacterial adherence was reduced to wild-type CHO cells treated with Lovastatin and to cholesterol-requiring insect cells grown in cholesterol-deficient medium, suggesting that the bacteria bound to plasma membrane cholesterol. Thus, this clinical isolate of Pseudomonas uses cholesterol and cholesterol esters for adherence. We propose that binding to cholesterol and cholesterol esters may affect the pathogenicity of similar Pseudomonas strains.