The presence of ACC and other mutations at codon 315 in the katG gene was detected by PCR amplification followed by restriction fragment length polymorphism (PCR-RFLP) generated with restriction enzymes Msp I and MspA1 I in 37 isoniazid-resistant and 22-susceptible Mycobacterium tuberculosis isolates from Kuwait obtained in 2001. The mutation AGC to ACC was detected in 22 (60%) isolates while any mutation at codon 315 of the katG gene was present in 24 (65%) of 37 isoniazid-resistant isolates. The typing studies showed that majority of the isolates carrying mutations at codon 315 exhibited unique DNA banding patterns. The results were extended by additional analysis of 67, 28 and 17 isoniazid-resistant and 18, seven and six-susceptible M. tuberculosis isolates from Kuwait, Dubai and Beirut, respectively, that were analyzed previously for ACC mutation alone. These studies showed that one of 21, one of 10 and two of 11 isolates (all recovered from patients of Middle Eastern origin) with no AGC to ACC mutation from Kuwait, Dubai and Beirut, respectively, contained other mutations at codon 315 of the katG gene. None of the susceptible strains contained any mutation at codon 315. The PCR-RFLP with MspA1 I that detects all mutations at codon 315, compared with Msp I that detects only ACC mutation, identified more isoniazid-resistant strains with mutations at codon 315 in the katG gene. The data also showed that mutations other than AGC to ACC at codon 315 in the katG gene occur frequently in M. tuberculosis isolates recovered from Middle Eastern patients and should be incorporated in a rapid screen for the detection of mutations for isoniazid-resistance in the katG gene from this ethnic group.