Most adenoviruses use the coxsackie-adenovirus receptor (CAR) as a major cellular receptor. We have shown recently that adenovirus types 8, 19a, and 37, which are the major causes of epidemic keratoconjunctivitis, use sialic acid rather than CAR as a major cellular receptor. The predicted isoelectric point of the receptor-interacting knob domain in the adenovirus fiber protein is unusually high (9.0-9.1) in type 8, 19a, and 37. The pKa of sialic acid is low, 2.6, implying a possible involvement of charge in fiber knob-sialic acid interactions. Here we show that (i) positively charged adenovirus knobs require sialic acid for efficient cell membrane interactions; (ii) viral and knob interactions with immobilized sialic acid or cell-surface sialic acid are sensitive to increased ionic strength; (iii) negatively charged molecules such as sulfated glycosaminoglycans inhibit the binding of virions to target cells in a nonspecific, charge-dependent manner; and that (iv) the ability of adenovirus knobs to interact with sialic acid correlates with the overall charge on the top surface of the respective knobs as predicted by homology modeling. Taken together, the results presented provide strong evidence for a charge mechanism during the interaction between the Ad37 fiber knob and sialic acid.