Numerous single nucleotide polymorphisms (SNPs) have been identified in the human genome, yet the functional significance of most is unknown. CYP3A4 is a key enzyme in the metabolism of numerous compounds. An A-->G substitution 290 bp upstream of the CYP3A4 transcription start site (CYP3A41B) has been associated with cancer phenotypes, but its phenotypic effects are unclear. To investigate the functional significance of CYP3A41B, we generated two luciferase reporter constructs: 1-kb (denoted L, long) and 0.5-kb (denoted S, short) promoter fragments containing either the variant (V(L),V(S)) or the wild-type (W(L), W(S)) sequences. We evaluated the effect of the variant sequence in the HepG2 and MCF-7 cell lines, and in primary human hepatocytes from three donors. Reporter constructs with the variant sequence had 1.2- to 1.9-fold higher luciferase activity than constructs with wild-type sequence in the cell lines (P < 0.0001) and hepatocytes (P = 0.021, P = 0.027, P = 0.061). The ratio of transcriptional activity for V(S):W(S) was similar to the V(L):W(L) ratio in HepG2 cells, but the V(S):W(S) ratio was consistently less than the V(L):W(L) ratio in MCF-7 cells. This suggests that CYP3A4 expression is higher from the variant promoter and that a repressor sequence may exist in the longer constructs. Electrophoretic mobility shift assays demonstrated specific binding of a component of HepG2 nuclear extract to both wild-type and variant promoters with consistently higher binding affinities to the wild-type sequence. This suggests the existence of a transcriptional repressor responsible for the lower CYP3A41A activity. Therefore, the phenotypic effects of the variant CYP3A41B may be associated with enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor.