The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified manual DNA extraction was evaluated for accuracy and ease of use. The sensitivity of LAMP in smear- and culture-positive sputum specimens was 97.7% (173/177 specimens; 95% confidence interval [CI], 95.5 to 99.9%), and the sensitivity in smear-negative, culture-positive specimens was 48.8% (21/43 specimens; CI, 33.9 to 63.7%). The specificity in culture-negative samples was 99% (500/505 specimens; CI, 98.1 to 99.9%). The average hands-on time for testing six samples and two controls was 54 min, similar to that of sputum smear microscopy. The optimal amplification time was 40 min. No indeterminate results were reported, and the interreader variability was 0.4%. Despite the use of a single room without biosafety cabinets for all procedures, no DNA contamination was observed. The assay was robust, with high end-point stability and low rates of test failure. Technicians with no prior molecular experience easily performed the assay after 1 week of training, and opportunities for further simplification of the assay were identified. Copyright © 2007, American Society for Microbiology. All Rights Reserved.