The use of co-trimoxazole in HIV-positive patients has been associated with a high frequency (40-80%) of hypersensitivity reactions. This has been attributed to the bioactivation of the sulphonamide component, sulphamethoxazole (SMX), to its toxic hydroxylamine and nitroso metabolites. The aim of this study was to determine whether functionally significant polymorphisms in the genes coding for enzymes involved in SMX metabolism influence susceptibility to SMX hypersensitivity. HIV-positive patients with (n = 56) and without (n = 89) SMX hypersensitivity were genotyped for allelic variants in CYP2C9, GSTM1, GSTT1, GSTP1 and NAT2 using polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymorphism analysis. The CYP2C9*2/*3 genotype and CYP2C9*3 allele frequencies were nine- and 2.5-fold higher in the hypersensitive group compared to non-sensitive patients, respectively, although they were not statistically significant when corrected for multiple testing. There were no differences in the frequencies of the GSTM1 and GSTT1 null genotypes, and the slow acetylator genotype, between hypersensitive and non-sensitive patients, while GSTP1 frequency was lower (although non-significant) in the hypersensitive group [21% versus 32%, odds ratio (OR) = 0.5, Pc = 0.24]. Comparison of the genotype frequencies in HIV-positive and -negative patients showed that the NAT2 slow acetylator genotype frequency in the HIV-positive patients (74%) was significantly (Pc = 0.0003, OR = 2.3) higher than in control subjects (56%). Our results show that genetic polymorphisms in drug metabolizing enzymes are unlikely to be major predisposing factors in determining individual susceptibility to co-trimoxazole hypersensitivity in HIV-positive patients.