Indirect evidence suggests that induced sputum derives from the surfaces of the bronchial airways. To confirm this experimentally, we employed a radiolabeled aerosol bolus delivery technique that preferentially deposits aerosol in the central airways in humans. We hypothesized that there would be significantly more radioactivity recovered in an induced sputum sample, and greater airways clearance of radiolabeled particles, immediately after a central versus peripheral airways deposition. Ten healthy volunteers underwent radiolabeled aerosol deposition ((99m)Tc sulfur colloid particles) to the central and peripheral airways on separate occasions followed immediately by induced sputum or no sputum (control), while seated in front of a gamma camera. Radioactivity was measured in the selected sputum sample, processed cell pellet, and supernatant fraction. Significantly more radioactivity was present in all portions of the sputum sample after central versus peripheral airways deposition (i.e., selected sample: 15,607 counts +/- 2,985 versus 943 counts +/- 298, p = 0.001). Clearance from the whole lung was significantly greater 40 min after central versus peripheral airways deposition (48 +/- 3% versus 5 +/- 1%, p = 0.0001). Compared with control, induced sputum greatly enhanced clearance after central deposition (48 +/- 3% versus 11 +/- 6%, p = 0.0001), but not after peripheral deposition (5 +/- 1% versus 3 +/- 0.8%). These results provide direct evidence that induced sputum derives from the central airways with little or no contribution from the peripheral airways.