STUDY OBJECTIVES Mycobacterium tuberculosis (MTb) bacilli are carried on airborne droplet nuclei produced by aerosolization that can occur from coughing, talking, or even singing. Because of their prolonged period of suspension, they can be filtered from the air onto a porous medium and readily detected using polymerase chain reaction (PCR). DESIGN Prospective cohort analysis. SETTING Samples of circulating air were collected over a 12-month period from within the rooms of 10 hospitalized patients who were under respiratory isolation to rule out MTb infection. A small laboratory pump was used to draw ambient air at a rate of 2 L/min over a 6-h period through a 0.2-microm polycarbonate membrane filter placed near the patient's bed. Analysis of the membrane filters was conducted using PCR. Sputum cultures for MTb were performed simultaneously, and the results of smears stained for acid-fast bacilli (AFB) were noted. MEASUREMENTS AND RESULTS MTb complex was successfully detected by PCR in six of seven patients in whom sputum MTb cultures were subsequently positive, and in zero of three with subsequently negative sputum cultures. Sampling in one patient with a positive culture, in whom PCR results were negative, was only carried out for 2 h due to pump malfunction. One of the six PCR-positive patients was AFB-smear negative at the time of air sampling. CONCLUSIONS Our preliminary findings indicate that the technique of Micropore membrane air sampling with PCR analysis has important applications in the epidemiology and diagnosis of MTb.