Pleural adenosine deaminase activity (ADA) is a sensitive and specific test for tuberculous pleurisy. Here, we report a case of undetectable ADA in the pleural fluid of a man presenting with chronic cough, fever and night sweats. Subsequent laboratory investigations and review revealed that the presence of high concentrations of ammonia in pleural fluid, commonly seen in empyema, negatively interferes with ADA results when measured by the Guisti and Galanti method. The source of ammonia may come from deamination of amino acids, ammonia-producing microbes and/or leucocytes. This interference invalidates ADA results and is present in ∼2% of our laboratory requests. It is important to keep this interference in mind when tuberculosis/ammonia-producing bacteria coinfections are suspected and during early phases of tuberculous pleurisy, when neutrophils predominate in the pleural fluids.