Identification of a hepatitis A virus (HAV) receptor is important for understanding HAV tissue tropism and replication sites and in the design of vaccines and antiviral therapy. The attachment of HAV to cultured cell lines was evaluated: Calcium-dependent specific attachment of four HAV strains to permissive cells occurred, whereas binding to nonpermissive cells did not. Investigation of HAV antigenic variant strains (neutralization escape mutants) demonstrated identical attachment properties with neutralization-susceptible strains, suggesting that the immunodominant neutralization antigenic site of HAV is not directly involved in cell attachment. Unlike foot-and-mouth-disease virus, a related picornavirus, RGD peptides (arginine-glycine-aspartic acid) were unable to interfere with HAV attachment. These studies demonstrate that HAV has a calcium-dependent receptor on cultured cell lines and suggest that the HAV binding region does not involve an RGD sequence or the HAV immunodominant neutralization site.