The T-786C promoter and 27-bp repeat intron 4 polymorphisms in the endothelial NO synthase (eNOS) gene have been inconsistently associated with various eNOS-related phenotypic changes. We explored molecular mechanisms underlying the inconsistency. We constructed pGL3 luciferase reporter vectors by inserting an eNOS promoter fragment containing either T or C nucleotide at -786 bp at the 5' end of the luciferase coding region and eNOS intron 4 containing either 5x or 4x27-bp repeats at the 3' end of the luciferase gene. The transcription efficiency in the T promoter was lower than in the C promoter (15.7+/-1.0% vs 83.3+/-5.8%, P<0.01 when 5x27-bp was an enhancer and 37.6+/-4.7% vs 58.9+/-7.5%, P<0.01 when 4x27bp was an enhancer). Although cigarette smoking extracts treatment increased the transcription efficiency significantly in the T promoter (1.7-fold, P<0.01), it reduced the C promoter efficiency (by 10% to 15%). A mobility shift assay revealed positive binding of the 27-bp repeat fragment with endothelial cell nuclear protein extracts. Our study demonstrates a cis-acting role of the 27-bp repeats in eNOS promoter function and a haplotype-specific expression pattern determined by DNA variants at -786 bp and intron 4 of the eNOS gene that is also modifiable by cigarette smoking.