Enviroxime and related analogs are potent inhibitors of rhinoviruses and enteroviruses in cell culture. Previous analyses of resistant mutants implicated the viral nonstructural protein 3A(B) as the likely target of drug activity. In this study, we used site-directed mutagenesis and selection of spontaneous rhinovirus 14 mutants with several enviroxime analogs to confirm the link between domains in rhinovirus 14 3A(B) and the function blocked by enviroxime. We also produced recombinant 3A and 3AB proteins for biochemical analyses. Despite extensive efforts, however, we were unable to demonstrate direct binding between enviroxime and any of several viral proteins, nor could we demonstrate binding of enviroxime to a host protein. In addition, enviroxime did not disrupt 3AB\'s ability to bind RNA or 3D polypeptide, the association of 3AB with membranes, or the cleavage of 3AB by 3C protease. Finally, we identified an enviroxime-resistant mutant with an increased level of resistance which apparently has mutations in multiple proteins or RNA sequences. Taken together, these results suggest that enviroxime targets a complex of proteins and/or cellular factors. Such a complex mechanism of inhibition might explain the low levels of viral resistance to these inhibitors as compared with other picornaviral inhibitors.