We have identified three new human tumor necrosis factor-α (TNF-α) promoter polymorphisms with single nucleotide (nt) substitutions at −862, −856, and −574 nt relative to the TNF-α transcription start site. The −862 and −856 nt TNF-α promoter polymorphisms occur with high frequency in Caucasian and Cambodian individuals and are each non-randomly associated with three extended HLA haplotypes. This study, in which 61 independent TNF-α promoters were analyzed spanning from −977 to +93 nt relative to the TNF-α mRNA cap site, establishes a new canonical TNF-α promoter sequence. Furthermore, we show that none of the three novel polymorphisms at −862, −856 and −574 nt or polymorphisms previously described at positions −238, −308 and +70 have an effect upon TNF-α gene expression in activated lymphocytes. Thus, these TNF-α promoter polymorphisms likely serve as markers for neighboring genes encoding HLA or other undefined molecules in the MHC that may influence disease susceptibility.