Protease activity is associated with the coadhesion of Actinomyces viscosus and Porphyromonas gingivalis. To try to distinguish whether the recognition/adhesion or degradative functions of proteases are more crucial for coadhesion, we determined the effect of trypsin and other purchased proteases and proteins on coadhesion when they were incorporated in the coadhesion assay buffer or when A. viscosus cells were pretreated with trypsin. Coadhesion was measured by the decrease in turbidity caused by the absorption of A. viscosus cells from aqueous suspension by P. gingivalis-coated hexadecane droplets. Pretreatment of A. viscosus with trypsin had no obvious effect on the kinetics of coadhesion. Likewise, trypsinization of A. viscosus failed to aid or enhance coaggregation by chemically induced, trypsin activity-deficient mutants of B. gingivalis. In contrast, incorporating trypsin in the buffer during the coadhesion assay yielded a concentration-dependent inhibition of coadhesion greater than the inhibition found with the same concentration of other proteases. Coadhesion was also impaired to a greater extent by similar wt/vol concentrations of nonproteolytic proteins (bovine serum albumin (BSA), defatted BSA, gelatin, and casein), by antisera against whole P. gingivalis cells and fimbriae, by preimmune serum, and by the amino acid arginine but not lysine. These findings suggest that the role of proteases in coadhesion is not solely to enzymatically "prime" A. viscosus for more avid coadhesion and that their role as potential protein or peptide seeking adhesins should be considered.