HLA-B27 gene, one of the HLA Class I molecule, is strongly associated with ankylosing spondylitis. It has been most frequently used as a disease-correlated HLA gene by clinicians. In most laboratories, conventional HLA-B27 typing is still performed by cell cytotoxicity test or fluorescence serology with specific antibodies. These methods, however, are labor intensive and take high regeant cost. In this study, more convenient and rapid DNA typing method for the determination of HLA-B27 gene was developed by using Polymerase Chain Reaction (PCR) technique. Genomic DNAs were extracted by phenol/chloroform procedure and amplified with B-27 allele specific primers. Four HLA-B27 cell lines (HOM-2, JESTHOM, WT24 and BTB) were used for the control experiment. The results of control cell and B-27 positive samples were correlated well with the data which was performed by aerological method. All of B27 positive PCR products were confirmed on southern blot hybridization using biotinylated probe. This study shows that the PCR technique is a practical tool than serological method for the determination of the HLA-B27 gene in routine clinical laboratory work.