A high affinity octamer transcription factor (OTF-1) binding site has been identified and characterized at position--46 base pairs (bp) in the proximal human lipoprotein lipase (LPL) promoter. The affinity of the LPL OTF-1 binding site was approximately 15-fold greater than a consensus octamer sequence, ATTTGCAT, present at position--66 bp in the mouse Vk T1 promoter, and approximately 5-fold greater than the OTF-1 site present at position--49 bp in the human histone H2B promoter. Diethylpyrocarbonate interference assays have identified both 5' and 3' adenine nucleotides, which flank the core LPL ATTTGCAT sequence and interfere with OTF-1 binding when chemically modified. Introduction of mutations in either 5' or 3' flanking AT-rich sequences lowered the affinity of OTF-1 binding below the level observed with the wild-type LPL octamer oligomer. A double mutation in both flanking AT regions, however, greatly reduced the affinity of this site to levels similar to that observed with the mouse Vk T1 OTF site. An additional nuclear transcription factor, NF-Y, has been shown to bind to a functional CCAAT box motif located at -65 bp in the LPL promoter using specific alpha-NF-Y antisera. The observation of high affinity OTF-1 and NF-Y binding sites in a region of the proximal LPL promoter which is necessary for high levels of LPL transcription suggests that these sites with their associated proteins play important functional roles in the transcriptional activation of the LPL promoter during adipocyte differentiation.