Decay accelerating factor (DAF) functions as a cell attachment receptor for a wide range of human enteroviruses, the interaction accounting for the haemagglutination phenotype exhibited by many members of this family. Haemagglutination inhibition assays using purified truncated soluble DAF (sDAF) receptors and short consensus repeat (SCR) domain-specific antibodies have been used to determine the domain(s) of DAF to which the viruses bind. Further sDAF-mediated virus neutralization and biosensor analysis have been used to confirm the virus-binding domains of DAF. Of the four distinct clusters of human enteroviruses, three contain representatives that bind DAF. The majority of DAF-binding enteroviruses occupy the 'CBV-like' cluster, and require SCR domains 2-4 for DAF binding. In contrast, the DAF-binding representatives of the 'ENV70-like' and 'PV-like' clusters require SCR1 for DAF interaction. These studies confirm that DAF binding is a widespread characteristic amongst phylogenetically divergent clusters within the enteroviruses and suggest that the ability to bind DAF may have evolved more than once within this group of viruses.