Infants with respiratory syncytial virus (RSV) infection were shown to have antibodies against HEp-2 cell antigen present in RSV-antigen preparation used for immunoblot analysis. The prevalence of anti HEp-2 cell antibodies was examined in infants hospitalized for RSV infection (n = 49, median age 121 days) compared to rotavirus infected children (n = 30, median age 114 days) and to healthy controls (n = 20, median age 150 days). The immunoblot analysis with RSV-infected and non-infected HEp-2 cells as antigen revealed the expected age-dependent low prevalence of G protein antibodies and clear seroconversion of N and P protein antibodies. HEp-2 antibody prevalence was higher in RSV antigen-positive infants (33/49) than in rotavirus antigen-positive (5/30) and RSV antigen-negative infants (4/20), respectively (p < 0.001). Anti HEp-2 antibodies were mostly directed against 47, 46, 33, 30 kD antigens. A multiple regression analysis found the following correlations (odds ratio; 95% confidence interval): 42 kD RSV antibodies (N protein) with pneumonia (7.58; 1.43-40), 94 kD RSV antibodies (G protein) with bronchiolitis (0.064; 0.006-0.686). This study shows repeated well-known features of humoral immunity in RSV infection. The data on anti HEp-2 antibodies point to a role for these pre-existing autoreactive antibodies in the pathogenesis of RSV infection.