The adherence properties of Pseudomonas aeruginosa strains with known pilin DNA sequences were studied. A polar pilus clearly contributed to adherence, as 35S-labeled pilus-positive (Pil+) strains bound significantly more to bovine trachea epithelial monolayers than did pilus-negative (Pil-) mutants (P less than .05) and minimally more than hyperpiliated strains. A pil- mutant PAK/NP, constructed by gene replacement, demonstrated low levels of attachment (3.8% of the inoculum adherent compared with 7% for the wild-type strain PAK), suggesting that other adhesins are functional. The Pil-, flagellum-negative strain PAO1150.1 bound the least (0.3% of the inoculum adherent), confirming the importance of motility in the binding process. The pilin sequences of strains P1 and PA1244 were virtually identical, although P1 bound threefold more than did PA1244. P1 produced 10-fold more proteinase than did PA1244, and a proteinase-negative mutant of P1, isolated by transposon mutagenesis, had binding equivalent to that of PA1244. The adherence of PAO1 was increased 60% in the presence of bacterial supernatants from phosphate-limited cultures and correlated with phospholipase C activity in the supernatant. Thus, the expression of several Pseudomonas genes may be required to promote efficient binding to epithelial surfaces.