Enviroxime is an antiviral compound that inhibits the replication of rhinoviruses and enteroviruses. We have explored the mechanism of action of enviroxime by using poliovirus type 1 and human rhinovirus type 14 as model systems. By varying the time of drug addition to virus-infected cells, we determined that enviroxime could be added several hours postinfection without significant loss of inhibition. This suggested that the drug targeted a step involved in RNA replication or protein processing. To identify this target, we mapped 23 independent mutations in mutants that could multiply in the presence of 1 microgram of enviroxime per ml. Each of these mutants contained a single nucleotide substitution that altered one amino acid in the 3A coding region. Using oligonucleotide-directed mutagenesis of cDNA clones, we have confirmed that these single-amino-acid substitutions are sufficient to confer the resistance phenotype. In addition, we conducted two experiments to support the hypothesis that enviroxime inhibits a 3A function. First, we determined by dot blot analysis of RNA from poliovirus-infected cells that enviroxime preferentially inhibits synthesis of the viral plus strand. Second, we demonstrated that enviroxime inhibits the initiation of plus-strand RNA synthesis as measured by the addition of [32P]uridine to 3AB in poliovirus crude replication complexes. To our knowledge, this is the first evidence that 3A can be targeted by antiviral drugs. We anticipate that enviroxime will be a useful tool for investigating the natural function of the 3A protein.