Guanidine resistance (gr) mutations of foot-and-mouth disease virus were mapped by recombining pairs of temperature-sensitive mutants belonging to different subtypes. In each cross, one parent possessed a gr mutation. Recombinants were isolated by selection at the nonpermissive temperature and assayed for the ability to grow in the presence of guanidine. From the progeny of three crosses, four different types of recombinant were distinguished on the basis of protein composition and RNA fingerprint. The sequences of the RNase T1-resistant oligonucleotides were determined and located in the full-length sequence of foot-and-mouth disease virus. The resulting maps show that (i) each recombinant was generated by a single genetic crossover, and (ii) both of the gr mutations studied were located within an internal 2.9-kilobase region which spans the P34 gene. This supports our hypothesis that guanidine inhibits the growth of foot-and-mouth disease virus by acting on nonstructural polypeptide P34. Additional evidence was provided by RNA fingerprinting gr mutants. In two of four cases the gr mutation was associated with a change in an oligonucleotide located near the 3' end of the P34 gene; in one of these the nucleotide substitution was identified.