Background: Determination of adenosine deaminase (ADA) activity is one of the most promising markers in diagnosing of tuberculous pleural effusion. ADA has two main isoenzymes: ADA1 and ADA2.The ADA2 is the predominant isoform in tuberculous pleural effusion, suggesting its important role as a diagnostic marker. This study was conducted to determine the diagnostic value of ADA and ADA2 measurement in tuberculous pleural effusion. Materials and Methods: Total ADA and ADA2 isoenzyme activities were measured in 93 case of pleural effusion, including tuberculosis (26males/5females), malignancy (22males/8females), empyema and para-pneumonic (11males/4females), transudate (6males/4females), rheumatoid arthritis and idiopathic (4males/3females). ADA levels were determined by Giusti and Galanti methods. ADA2 was measured with a potent inhibitor of ADA1 isoenzyme. Results: Total ADA and ADA2 activities in tuberculous exudates were 96.6±29.1 and 74.4±29 U/L, respectively. With diagnostic thresholds of 46 and 42 U/L, the sensitivities of ADA and ADA2 for tuberculous exudates were 100% and 97%; their specificities 82 and 88%; and their efficiencies 88% and 93.5%, respectively. All tuberculous exudates, 2 neoplastic, 8 para- infective (including 4 empyemas) and one rheumatoid arthritis had total ADA levels >46 U/L; of these, only one lymphoma and one rheumatoid arthritis had ADA2/ADA activity ratio >50%. Considering simultaneous criteria of total ADA more than 46U/L, ADA2 >42 U/L and ADA2/ADA more than 50%, we had only two false positive results, rising the specificity up to 96%. Conclusion: 1. ADA2 is a more efficient diagnostic marker for Tuberculous pleural effusion compared with total ADA. 2. Overall, diagnostic value of ADA would be enhanced by the determination of its isoenzymes, especially for distinguishing between the tuberculous and para-infective effusions. (Tanaffos 2005; 4(15): 37-42)