Objectives: Efavirenz is widely prescribed for HIV-1 infection, and CYP2B6 polymorphisms 516G →Tand 983T→C define efavirenz slow metabolizer genotypes. To identify genetic predictors of higher plasma efavirenz concentrations beyond these two common functional alleles, we characterized associations with mid-dosing interval efavirenz concentrations in 84 HIV-infected adults, all carrying two copies of these major loss-of-function CYP2B6 alleles. Methods: Study participants had been randomized to efavirenz-containing regimens in prospective clinical trials and had available plasma efavirenz assay data. Analyses focused on secondary metabolism pathway polymorphisms CYP2A6 -48T→G (rs28399433), UGT2B7 735A→G (rs28365062) and UGT2B7 802T→C (rs7439366). Exploratory analyses also considered 196 polymorphisms and 8 copy number variants in 41 drug metabolism/transport genes. Mid-dosing interval efavirenz concentrations at steady-state were obtained ≥8 h but,19 h post-dose. Linear regression was used to test for associations between polymorphisms and log-transformed efavirenz concentrations. Results: Increased efavirenz concentrations were associated with CYP2A6 -48T→G in all subjects (P=3.8×10-4) and in Black subjects (P=0.027) and White subjects (P=0.0011) analysed separately; and with UGT2B7 735 G/G homozygosity in all subjects (P=0.006) and in Black subjects (P=0.046) and White subjects (P=0.062) analysed separately. In a multivariable model, CYP2A6 -48T→G and UGT2B7 735 G/G homozygosity remained significant (P,0.05 for each). No additional polymorphisms or copy number variants were significantly associated with efavirenz concentrations. Conclusions: Among individuals with a CYP2B6 slow metabolizer genotype, CYP2A6 and possibly UGT2B7 polymorphisms contribute to even higher efavirenz concentrations. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.