Human genome sequencing has resulted in a great body of data, including a stunningly large number of single nucleotide polymorphisms (SNPs) with unknown phenotypic manifestations. Identification and comprehensive analysis of regulatory SNPs in human gene promoters will help quantify the effects of these SNPs on human health. Based on our experimental and computer-aided study of SNPs in TATA boxes and the use of literature data, we have derived an equation for TBP/TATA equilibrium binding in three successive steps: TATA-binding protein (TBP) sliding along DNA due to their nonspecific affinity for each other ↔ recognition of the TATA box ↔ stabilization of the TBP/TATA complex. Using this equation, we have analyzed TATA boxes containing SNPs associated with human diseases and made in silico predictions of changes in TBP/TATA affinity. An electrophoretic mobility shift assay (EMSA)-based experimental study performed under the most standardized conditions demonstrates that the experimentally measured values are highly correlated with the predicted values: the coefficient of linear correlation, r, was 0.822 at a significance level of α<10⁻⁷ for equilibrium K(D) values, (-ln K(D)), and 0.785 at a significance level of α<10⁻³ for changes in equilibrium K(D) (δ) due to SNPs in the TATA boxes (δ= -ln[K(D,TATAMut)]-(-ln[K(D,TATAMut)])). It has been demonstrated that the SNPs associated with increased risk of human diseases such as α-, β- and δ-thalassemia, myocardial infarction and thrombophlebitis, changes in immune response, amyotrophic lateral sclerosis, lung cancer and hemophilia B Leyden cause 2-4-fold changes in TBP/TATA affinity in most cases. The results obtained strongly suggest that the TBP/TATA equilibrium binding equation derived can be used for analysis of TATA-box sequences and identification of SNPs with a potential of being functionally important.