We investigated the mechanisms by which respiratory syncytial virus (RSV) infection decreases vectorial Na+ transport across respiratory epithelial cells. Mouse tracheal epithelial (MTE) cells from either BALB/c or C57BL/6 mice and human airway H441 cells were grown on semipermeable supports under an air-liquid interface. Cells were infected with RSV-A2 and mounted in Ussing chambers for measurements of short-circuit currents (Isc). Infection with RSV for 24 hours (multiplicity of infection = 1) resulted in positive immunofluorescence for RSV antigen in less than 10% of MTE or H441 cells. In spite of the limited number of cells infected, RSV reduced both basal and amiloride-sensitive Isc in both MTE and H441 cells by approximately 50%, without causing a concomitant reduction in transepithelial resistance. Agents that increased intracellular cAMP (forskolin, cpt-CAMP, and IBMX) increased mainly Cl- secretion in MTE cells and Na+ absorption in H441 cells. RSV infection for 24 hours blunted both variables. In contrast, ouabain sensitive Isc, measured across apically permeabilized H441 monolayers, remained unchanged. Western blot analysis of H441 cell lysates demonstrated reductions in α- but not γ-ENaC subunit protein levels at 24 hours after RSV infection. The reduction in amiloride-sensitive Isc in H441 cells was prevented by pretreatment with inhibitors of de novo pyrimidine or purine synthesis (A77-1726 and 6-MP, respectively, 50 μM). Our results suggest that infection of both murine and human respiratory epithelial cells with RSV inhibits vectorial Na+ transport via nucleotide release. These findings are consistent with our previous studies showing reduced alveolar fluid clearance after RSV infection of BALB/c mice.