Understanding transcription initiation of the endothelial nitric-oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present study therefore focused upon a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of constitutive expression of the human eNOS gene in vascular endothelium. Two tightly clustered cis-regulatory regions were identified in the proximal enhancer of the TATA-less eNOS promoter using deletion analysis and linker-scanning mutagenesis: positive regulatory domains I (-104/-95 relative to transcription initiation) and II (-144/-115). Analysis of trans-factor binding and functional expression studies revealed a surprising degree of cooperativity and complexity. The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional domain studies in Drosophila Schneider cells and endothelial cells revealed examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ. Therefore, multiprotein complexes are formed on the activator recognition sites within this 50-base pair region of the human eNOS promoter in vascular endothelium.