The goals of these experiments were to efficiently deliver aerosolized adeno-associated virus (AAV) vector to the lungs of Rhesus macaques and to measure gene transfer and expression. To determine optimal lung deposition, we compared four techniques of delivering aerosolized saline admixed with the radioisotope (99m)technetium ((99m)Tc) nebulized through a mouthpiece (Neb Oral), a laryngeal airway mask (Neb LMA), or an endotracheal tube (Neb ETT), or bronchoscopically delivered by Microsprayer (PennCentury). Total lung deposition fraction, as indicated by gamma scintigraphy, averaged 0.5% (Neb Oral), 1.2% (Neb LMA), 1.8+/-0.4% (Neb ETT), and 62.3+/-11.3% (Microsprayer). Because microspraying was the most efficient method of delivery, we used it to administer saline with (99m)Tc-labeled diethylene-triamine penta-acetic acid (DTPA) admixed with 9 x 10(11) infectious units (i.u.) of AAV serotype 2 (rAAV2) vector encoding green fluorescent protein (GFP; rAAV2-GFP). Initial total and regional lung depositions were quantified by scintigraphy. We analyzed the tissue three weeks later for vector-specific DNA transduction and RNA expression. Radioisotope was detected in all lung regions, reflecting an average dose of 1.33 x 10(10)+/-9.5 x 10(9) i.u. per region. Regional data indicated an increase in expression when the dose exceeded 3 x 10(9) i.u. (P=0.030). We conclude that expression of rAAV2-GFP in lungs appears to be related to depositing a regional threshold dose greater than 3 x 10(9) i.u., easily achieved by bronchoscopic microspraying.