The interaction of the macrophage cell line P388D1 with Pseudomonas aeruginosa in the absence of stimulators or opsonins led to substantial association of bacteria, as judged by visual counting and FACScan assays. This association was observable within 5 min of addition of bacteria, could not be disturbed by exhaustive washing, and occurred with pilus- or flagellum-deficient mutants but not with rpoN mutants, which have been proposed to lack a secondary adhesin. In contrast, specific antibody was capable of causing similar enhancement of bacterial uptake regardless of the rpoN phenotype. Fibronectin stimulated uptake of bacteria with the pilus as an adhesin, and stimulation was observable within 5 min. Both fibronectin-enhanced and antibody-opsonized uptake were susceptible to inhibition by pertussis toxin but not by cholera toxin. The influence of fibronectin on P388D1 cells was distinguishable from that of lipopolysaccharide, which caused substantial morphological changes in cells. Although lipopolysaccharide stimulated bacterial uptake, it actually suppressed fibronectin-mediated enhancement of uptake at high concentrations.