Three ponies were inoculated with plasma containing 10(4.8) TCID50 of equine infectious anemia virus (EIAV) and observed for 165 to 440 days. Each pony developed a febrile response within 3 weeks of infection during which a plasma viremia greater than or equal to 10(3.5) TCID50/ml was observed. Analyses of four isolates from sequential febrile episodes in a single pony were conducted by two-dimensional tryptic peptide maps and with monoclonal antibodies in immunoblots. Structural and antigenic alterations were observed in the envelope glycoproteins gp90 and gp45, with greatest variation in gp90. Specific IgG to EIAV gp90, gp45, and p26 of homologous and heterologous isolates was detectable by immunoblots within one month after infection although IgG levels to gp45 at this time were relatively low. The group-specific determinants of gp90 and gp45 were more antigenic than those of p26; however, binding of IgG to these determinants did not correlate with neutralization of EIAV as assayed in fetal equine kidney cells. Neutralizing antibodies were first detectable within two months of infection and only neutralized viruses isolated prior to serum collection. Neutralizing activity of sera collected later in the infection was broadly reactive regardless of the number of clinical episodes the donor had suffered.