To evaluate the efficacy of three sputum acid-fast bacillus (AFB) smears to rule out pulmonary tuberculosis, sputum AFB smear and culture results were analyzed at two university-affiliated teaching hospitals. The negative predictive value of the smear increased by only 0.2% on days 2 and 3 each, indicating that in low-prevalence populations, there is limited value in requiring three negative sputum AFB smears before discontinuing tuberculosis isolation.