Rotavirus NSP4, the first described viral enterotoxin, localizes to the plasma membrane of infected cells, possibly through interaction with caveolin-1. A direct interaction between NSP4 and caveolin-1, the structural protein of caveolae, was shown by yeast two-hybrid, peptide binding, and FRET analyses. To dissect the precise NSP4 binding domain to caveolin-1, mutants were prepared by altering either the charged or hydrophobic face of the NSP4 C-terminal amphipathic alpha-helix and examined for binding to caveolin-1. Replacing six charged residues with alanine (FLNSP4Ala) disrupted the charged face, while the hydrophobic face was disrupted by replacing selected hydrophobic residues with charged amino acids (aa) (FLNSP4HydroMut). In yeast two-hybrid and peptide binding assays, FLNSP4Ala retained its binding capacity, whereas FLNSP4HydroMut failed to bind caveolin-1. Mutants were generated with an Nterminal truncated clone (NSP446-175), which removed the hydrophobic domains and aided in yeast-two hybrid assays. These mutants exhibited the same binding pattern as FLNSP4 confirming that the N-terminus of NSP4 lacks the caveolin-1 binding site and NSP446-175 is sufficient for binding. Seven additional mutants were prepared from NSP4HydroMut in which individually charged residues were reverted to the original hydrophobic aa or were replaced with alanine. Analyses of the interaction of these revertants with caveolin-1 localized the NSP4 binding domain to one critical hydrophobic aa (L116) and one or two additional aa (I113, L127, and/or L134) on the hydrophobic face. Those mutants that bound caveolin-1 bound both the N- and C-terminal caveolin-1 peptides, but lacked binding to a centrally located peptide. These data suggest conformational and hydrophobic constraints play a role in the NSP4-caveolin-1 association. The mutant NSP4 molecules also were evaluated for transport to the plasma membrane. Mammalian cells were transfected with FLNSP4, FLNSP41-175Ala, and NSP41-175HydroMut plasmid DNA, surface biotinylated, and examined by IFA or Western blot for NSP4 expression. Epifluorescence revealed FLNSP4 and FLNSP4Ala were exposed on the cell surface in the absence of other viral proteins, whereas NSP4HydroMut remained intracellular. Further, NSP4-transfected cells displayed an intracellular association of with caveolin-1 or the caveolin-1 chaperone complex proteins. These data indicate NSP4 interacts with caveolin-1 in the absence of other viral proteins.