Gene therapy using rAd shows promise in treating genetic pulmonary diseases. Inherited deficiency of SP-B, a proteolipid critical to pulmonary surfactant, results in severe respiratory failure in term infants. SP-B is produced by alveolar type II cells and bronchiolar Clara cells and may be harmful within other cells due to its fusogenic properties. A SP-B promoter(SPB; -640/+319 relative to transcription start site) shows lung epithelial cell line specificity in transient transfection of H441 pulmonary adenocarcinoma cells with promoter strength similar to the promiscuous viral RSV promoter. To explore cell-type specific gene therapy for SP-B deficiency, we compared expression of the lacZ reporter gene driven by the RSV or SP-B promoters in rAd. Dose-response and time course experiments in H441 cells demonstrated efficient rAd.SPB.lacZ and rAd.RSV.lacZ infection with 24h of virus exposure at a multiplicity of infection (MOI) of≈1-10 with high-level β-galactosidase activity after 72h. X-gal staining showed specificity of rAd.SPB.lacZ for pulmonary derived cell lines (A549 and H441 versus HeLa) whereas all cell lines stained extensively with rAd.RSV.lacZ. This finding was confirmed by quantitative β-gal assay: the ratios of rAd.SPB.lacZ to rAd.RSV.lacZ β-gal activities(average ± SEM) were A549 = 1.96 ± 0.69; H441 = 0.66 ± 0.13; HeLa = 0.05 ± 0.01; p<0.05; n=3). Human fetal lung explants(24wk gestation) infected with rAd.SPB.lacZ had preferential X-gal staining of airway lining cells versus interstitial cells. Epithelia of explanted trachea showed minimal staining. All lung and trachea explant cell types stained extensively with rAd.RSV.lacZ. rAd.SPB.lacZ infection of mixed cells isolated from lung explants showed staining of most epithelial cells and rare fibroblasts. Primary fibroblasts infected with rAd.SPB.lacZ (MOI 1-300) had very low β-gal activities (7-94 A420/min/mg protein) whereas β-gal activities of fibroblasts infected with rAd.RSV.lacZ increased in a dose-dependent manner (480-55200 A420/min/mg protein). These results indicate that the SP-B promoter in rAd maintains cell line specificity and may be useful for limiting transgene expression to type II and Clara cells. (supported by 5 P30 HD28815)