A total of 183 epidemiologically unlinked Mycobacterium tuberculosis isolates collected in the St. Petersburg area of Russia from 1996 to 2001 were screened for alterations in codon 306 of the embB gene; mutations in this codon are reported to confer resistance to ethambutol (EMB). The embB306 mutations were detected in 14 (48.3%) of 29 EMB-resistant strains and, quite surprisingly, in 48 (31.2%) of 154 EMB-susceptible strains. A discrepancy between the results of phenotypic and genotypic EMB resistance tests was restricted to the strains already resistant to other antitubercular (anti-TB) drugs. In particular, 40 (60%) of the 69 EMB-susceptible strains resistant to rifampin, isoniazid, and streptomycin but none of the 43 pansusceptible strains harbored an embB306 mutation. We hypothesize that the phenomenon observed could reflect the presence of a target other than EmbB for the drug in tubercle bacilli; this unknown target could be sensitized and affected, sensu lato, by EMB during treatment with other first-line anti-TB drugs. Comparison with DNA fingerprinting data showed that, irrespectively of the phenotypic susceptibility profiles, 46 (50.6%) of 91 Beijing family strains and 16 (17.4%) of 92 strains of other genotypes had a mutation in embB306.