A great deal of evidence has shown that non-human leucocyte antigen (HLA)-B27 genes may play crucial roles in the aetiology of ankylosing spondylitis (AS), but there is little evidence of a relationship with tumour necrosis factor (TNF)alpha gene variation. One functional single-nucleotide polymorphism (SNP), -850 C-->T, on the TNFalpha gene promoter region was identified and confirmed to be significantly associated with AS by our recent study.To investigate whether the -850 C-->T SNP is a susceptibility locus for AS or is only a marker linked to potential disease gene loci in a Chinese population.Ten common SNPs were selected from nine inflammatory genes covering the right and left flanking regions of the TNFalpha gene, which span a region of about 100 kb on chromosome 6p21.31, and a tag SNP in HCP5 gene was used to examine the linkage between the HLA-B27 and TNFalpha genes. SNPs were genotyped by PCR restriction-fragment length polymorphism (RFLP), allele-specific PCR and restriction site-generating PCR-RFLP for single-base association and linkage disequilibrium (LD).The prevalence of TNFalpha-850 C-->T SNP was significantly different between case and control groups. A specific haplotype covering TNFalpha gene mutant was strongly associated with AS. An LD test showed that a recombination between HLA-B27 and TNFalpha might have taken place.The TNFalpha locus was reconfirmed and showed association with susceptibility to AS. It may be independent of HLA-B27. A range of 58 kb covering TNFalpha had strong LD to AS.