We prepared and characterized human monoclonal Fab fragments to rotavirus from IgG1 kappa combinatorial libraries (designated as N and O) constructed from peripheral blood lymphocytes (PBLs) from two healthy individuals. Approximately 30-fold enrichment in eluted phage was obtained in these libraries after five rounds of panning against rabbit polyclonal antibody-captured human rotavirus (HRV) Wa strain. Forty-eight clones from each library were tested for reactivity to HRV Wa in an enzyme-linked immunosorbent assay (ELISA), and the identities of positive clones were determined by BstNI fingerprinting. As a result, eight individual clones (five from N library and three from O library) were isolated. In testing the cross-reactivity of Fabs against a panel of self- or non-self antigens, all Fab clones were found to be specific for HRV Wa. Fab clones from the two libraries showed distinct characteristics with respect to their reaction patterns with Wa and crossreactivities with rotavirus strains, and displayed variable heavy (VH) chain gene usage, although they recognized the VP6 protein as determined by immunoblotting. The distinct epitope recognition by Fabs from two libraries suggests different courses of humoral immune response to rotavirus during infection in the two individuals.