The objectives of the this study were to assess the influence of CYP3A5 genotype and sex on the variability in total CYP3A activity and to compare 4b-hydroxycholesterol and omeprazole sulfoxidation as phenotypic markers for CYP3A activity in Ethiopians. Healthy subjects (n ¼ 150) were genotyped for CYP3A5*3, *6 and *7 using allele-specific PCR and Taqman genotyping assays. Plasma levels of 4b-hydroxycholesterol, 3 h post-dose omeprazole and omeprazole sulfone, were determined by gas chromatography-mass spectrometry and high performance liquid chromatography, respectively. The frequency of CYP3A5*1, *3, *6 and *7 was 20.5, 67.3, 12.2 and 0%, respectively. The mean plasma 4b-hydroxycholesterol level was 35.4 ng ml À1. The mean 4b-hydroxycholesterol level (P ¼ 0.0001) and the 4b-hydroxycholesterol/cholesterol ratio (P ¼ 0.004) were higher in women than in men. CYP3A5 genotype significantly correlated with the plasma 4b-hydroxycholesterol concentration (P ¼ 0.003) and 4b-hydroxycholesterol/ cholesterol ratio (P ¼ 0.0002). The omeprazole/omeprazole sulfone ratio was significantly correlated with 4b-hydroxycholesterol and 4b-hydroxycholesterol/cholesterol ratio in CYP3A5*0/*0 genotypes but not in individuals carrying the CYP3A5*1 allele. No correlation of omeprazole/omeprazole sulfone ratio with sex or CYP3A5 genotype was observed. A clear gene-dose effect implies plasma 4b-hydroxycholesterol level as a useful endogenous biomarker for total CYP3A activity (CYP3A5 plus CYP3A4) whereas the omeprazole/omeprazole sulfone ratio reflects mainly CYP3A4 activity. Sex and CYP3A5 genotype influence total CYP3A activity. Ethiopians display high total CYP3A activity and a unique distribution of CYP3A5 variant alleles not described hitherto.