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The human tumor necrosis factor-[Alpha] (TNF-[Alpha]) gene encodes a pleiotropic cytokine that plays a critical role in basic immunologic processes. To investigate the TNF-[Alpha] regulatory region in the primate lineage, we isolated TNF-[Alpha] promoters from representative great apes, Old World monkeys, and New World monkeys. We demonstrate that there is a nonuniform distribution of fixed human differences in the TNF-[Alpha] promoter. We define a 'fixed human difference' as a site that is not polymorphic in humans, but which differs in at least one of the seven primate sequences examined. Furthermore, we identify two human TNF-[Alpha] promoter single nucleotide polymorphisms that are putative ancestral polymorphisms, because each of the human polymorphic nucleotides was found at the identical site in at least one of the other primate sequences. Strikingly, the largest conserved region among the primate species, a 69-nt 'phylogenetic footprint,' corresponds to a region of the human TNF-[Alpha] promoter that forms the transcriptionally active nucleoprotein-DNA complex, essential for gene regulation. By contrast, other regions of the TNF-[Alpha] promoter, which exhibit a high density of variable sites, are nonessential for gene expression, indicating that distinct TNF-[Alpha] promoter regions have been subjected to different evolutionary constraints depending on their function. TNF-[Alpha] is the first case in which a promoter region dissected by functional analyses can be correlated with nucleotide polymorphism and variability in primate lineages. The results suggest that patterns of polymorphism and divergence are likely to be useful in identifying candidate regions important for gene regulation in other immune-response genes.