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The Human Major Histocompatibility Complex (MHC) plays a crucial role in transplantation, transfusion, paternity test and assessment of susceptibility to some diseases associated with HLA-B27. Three of the most fashionable methods for determination of HLA antigens in clinical and research laboratories are MICROLYMPHOCYTOTOXICITY (MLCT), FLOWCYTOMETRY and polymerase chain reaction (PCR). The purpose of this study was to compare the sensitivity, specificity, and positive and negative predictive values of MLCT and FLOWCYTOMETRY methods with PCR. as a gold standard method in determination of HLA-B27 antigen. In the present study, all three above-mentioned techniques have been used for 36 patients suffering from ANKYLOSING SPONDYLITIS and 31 healthy volunteers. Specific antisera and monoclonal antibodies against HLA-B27, and allele specific PCR. have been used in MLCT, FLOWCYTOMETRY and PCR. methods respectively. The results show that sensitivity, specificity, positive and negative predictive values of MLCT method as compared with PCR. technique were 83.3%, 100%, 100% and 88.1% respectively. Moreover, sensitivity, specificity, positive and negative predictive values of FLOWCYTOMETRY compared to the PCR. technique were 100%, 94.6%, 93.8% and 100% respectively. Based on the results, the FLOWCYTOMETRY method in determination of HLAB27 is more valid than MLCT in this regard, particularly in research programs. The similarity between the results of our study and those studies done in Europe suggests the probability of resemblance between HLA-B27 subtypes in Europe and in …