The aim of this study was to determine the prevalence of Ser315Thr substitution in isoniazid (INH)-resistant strains of Mycobacterium tuberculosis in Uruguay. The katG gene of 62 INH-resistant strains was analysed by an RFLP-PCR assay. PCR products were digested with MspI to detect Ser315Thr and Arg463Leu substitutions. A total of 16 of the 62 (26 %) INH-resistant strains analysed had a Ser315Thr substitution. Only one INH-resistant strain had an Arg463Leu substitution and two strains had a deletion in katG. Of the 16 strains with Ser315Thr, 15 showed different profiles using a double-repetitive-element PCR assay, demonstrating that there was no local dissemination of any particular strain. These findings are in agreement with published data from regions where the prevalence of tuberculosis (TB) is intermediate and may be due in part to the success of the local TB control programme.