Absence of a maximal dose-response plateau and mathematical modeling suggest that asthmatic airways close during bronchoconstriction. Finding segmental areas affected by closure would be important in understanding asthmatic airway function. The aim of this study was to evaluate single-photon emission computed tomography (SPECT) as a method of investigating airway closure. Simultaneous SPECT transmission and emission studies were performed on a thoracic phantom to develop analysis methodology, and on 13 normal subjects after they inhaled a Technegas bolus from residual volume (RV), to measure airway closure. Single-breath nitrogen test values and lung volumes were measured. Airway closure was defined as the percent of Technegas-free lung volume (LVclosed). The mean error +/- 95% CI of the error, as determined by transmission scan, was 1.1 ml +/- 165 ml (0.8% +/- 15% lung volume) in the phantom studies, and 112 ml +/- 419 ml (4% +/- 31% of supine functional residual capacity [FRC]) in the human studies. LVclosed correlated with closing capacity (r = 0.86, p < 0.01 ) and closing volume (r = 0.86, p < 0.01), but not with RV/total lung capacity (TLC). This study indicates that simultaneous SPECT emission and transmission scans, using a Technegas bolus, are a valid method of measuring airway closure in vivo, with the added advantage of providing three-dimensional data that allow the detection of small, discrete areas of airway closure and determination of their volumes and shapes.