Glutaraldehyde (GTA) is a potent virucidal disinfectant whose exact mode of action against enteroviruses is not understood. Earlier reports showed that GTA reacts preferentially with the VP1 capsid protein of echovirus 25 and poliovirus 1 and that GTA has affinity for exposed lysine residues on proteins. To investigate further the inactivation of enteroviruses by GTA, seven strains were selected on the basis of differences in their overall number and the positions of lysine residues in the amino acid sequences of the VP1 polypeptide. Inactivation kinetics experiments were performed with 0.10% GTA. The viruses grouped into three clusters and exhibited significantly different levels of sensitivity to GTA. The results were analyzed in the light of current knowledge of the three-dimensional structure of enteroviruses and the viral life cycle. The differences observed in sensitivity to GTA were related to the number of lysine residues and their locations in the VP1 protein. The overall findings suggest that the BC and DE loops, which cluster at the fivefold axis of symmetry and are the most exposed on the outer surface of the virions, are primary reactive sites for GTA.