Thirty Japanese clinical isolates of Mycobacterium tuberculosis were analysed by pyrazinamide susceptibility testing and pyrazinamidase assay, as well as polymerase chain reaction for single-strand conformational polymorphism and direct sequencing of the gene encoding pyrazinamidase (pncA). All sensitive isolates showed pyrazinamidase activity and a wild-type pncA gene, but three resistant isolates had pncA gene mutations and lacked pyrazinamidase activity. The latter isolates showed a minimum inhibitory concentration of at least 100 mg/l by the 7H10 agar proportion method and 400 mg/l by the 7H9 liquid medium method. Isolate 28 showed T-to-C change at position 11, leading to Leu4→Ser substitution; isolate 29 had an 8-bp deletion from position 382; and isolate 30 had A-to-C change at position 29, leading to Gln10→Pro substitution. The deletion has not been described previously. This is the first demonstration of pncA gene mutations in PZA-resistant M. tuberculosis strains isolated from Japanese patients.