The ability to predict drug deposition of inhaled drugs used in cystic fibrosis (CF) is important if there is a need to target specific doses of drug to the lungs of individual patients. The gold standard of measuring pulmonary deposition is the quantification of an aerosolized radiolabel either mixed with the drug solution or tagged directly to the compound of interest. Accuracy of the quantification could be assured if there is agreement between the amount of radioactivity before and after administration. Before administration, the radiolabel is concentrated in the well of the nebulizer, whereas after administration, it is distributed throughout the nebulizer, the expiratory filter and connectors, and the upper airway, stomach, trachea, and lung. Not only is the geometry of the distribution that is presented to the gamma camera different, but there are different attenuation factors for the various body tissues. The primary aim of this study was to evaluate the accuracy of the quantification of deposition. Secondary goals were to compare in vitro nebulizer performance with that measured in vivo during the deposition study. Eighty milligrams of tobramycin and technetium bound to human serum albumin was administered to 10 normal adults using a Pari LC Jet Plus (Pari Respiratory Equipment, Inc., Richmond, VA) breath-enhanced nebulizer. Techniques were developed that allowed for the accounting of 99 +/- 2% of the initial radioactivity. The fraction of the rate of lung deposition to total body deposition was the in vivo respirable fraction (0.62 +/- 0.07), which closely agreed with in vitro measurements of respirable fraction (0.62 +/- 0.04). Drug output measured from the change in weight and concentration in the nebulizer systematically overestimated drug output measured by the deposition study. The results indicate that 11.8 of the initial 80 mg would be deposited in the lungs. This technique could be adapted to accurately quantify the amount of deposition on any inhaled therapeutic agent, but caution must be used when extrapolating performance of a nebulizer on the bench to expected deposition in patients.