Rotavirus (RV) is the leading cause of infantile gastroenteritis worldwide. RV nonstructural protein 4 (NSP4), the first characterized viral enterotoxin, is a 28-kDa glycoprotein that has pleiotropic functions in RV infection and pathogenesis. NSP4 has multiple forms enabling it to perform its different functions. Dissecting such functions could be facilitated by use of epitope-specific antibodies. This work mapped the epitopes for the monoclonal antibody B4-2/55 and three polyclonal antisera generated against synthetic SA11 NSP4 peptides corresponding to residues 114-135, 120-147, and 150-175. The epitope for B4-2/55 mapped to residues 100-118, wherein residues E105, R108 and E111 are critical for antibody binding. Antiserum generated to two peptides (aa114-135 and aa120-147) with enterotoxin activity each recognize a single but distinct epitope. The epitope for the peptide antiserum to aa114-135 was mapped to residues 114-125 with highly conserved residues T117/T118, E120, and E122 being critical for antibody binding. The peptide antiserum to aa120-147 binds to NSP4 at residues 130-140 and residues Q137-T138 are critical for this epitope. Finally, the epitope for the antiserum to peptide aa150-175 mapped to residues 155-170, wherein residues E160 and E170 are critical for antibody binding. Knowledge of the binding sites of domain-specific antibodies can aid in further characterizing different functions of NSP4. To demonstrate this, we characterized the interaction between NSP4 and VP5() [K(D)=0.47 microM] and show that binding of NSP4 to VP5* is blocked by antibody to NSP4 aa114-135 and aa120-147, but not aa150-175. The use of single epitope-specific antibodies to differentially block functions of NSP4 is a feasible approach to determine the functional domain structure of this important RV virulence factor.