The location of tumor necrosis factor-alpha gene (TNFa) at a susceptibility locus for ankylosing spondylitis (AS) on chromosome 6p21.3, has been identified by several linkage studies across multiple population groups. In the light of these findings, we have carried out case-control studies in two independent Chinese AS cohorts (Shanghai, Fuzhou). Both have revealed a novel risk allele (-857T) with AS (best P = 5.12E-21 and 2.32E-11 for Shanghai population and Fuzhou population, respectively). Further association studies have suggested that -857T significantly increases the susceptibility to AS in both the HLA-B27-positive and HLA-B27-negative groups (global P = 0.0062 and 0.0065, respectively). The genotype-relative-risk analyses have indicated that the minor allele-containing (TT+TC) genotypes increase AS susceptibility to a greater degree than the homozygous-major-allele (CC) genotype frequency in cases of-857C>T (TT + CT vs. CC, P = 0.0092 and 0.0025 respectively, in the HLA-B27 positive and negative groups). Single-SNP analyses as well as haplotype analyses have revealed that -857C>T appears to be the most likely variants underlying the association of the TNFa region with AS independently of HLA-B27.