We investigated mechanisms underlying GATA-6-mediated transcriptional activation of the alveolar epithelial type I cell-enriched gene aquaporin-5 (AQP5). GATA-6 expression increases in alveolar epithelial cells in primary culture, concurrent with upregulation of AQP5 and transition to a type I cell-like phenotype. Co-transfections in MLE-15 and NIH3T3 cells demonstrated trans-activation by GATA-6 of a rat 1716-bp-AQP5-luciferase (-1716-AQP5-Luc) reporter. Electrophoretic mobility shift assay and chromatin immunoprecipitation identified an interaction between GATA-6 and putative binding sites in the AQP5 promoter. However, mutation of these sites did not reduce GATA-6-mediated activation, implicating mechanisms in addition to direct binding of GATA-6 to DNA. A 5'-deletion construct, -358-AQP5-Luc, which does not encompass GATA motifs, was still activated by GATA-6 by as much as 50% relative to -1716-AQP5-Luc. Internal deletion of the -358/-173 GC-rich domain, which includes several putative Sp1 consensus sites, reduced trans-activation by 60%, suggesting importance of this region for GATA-mediated activity. -358-AQP5-Luc was similarly activated by both GATA-6 and a GATA DNA binding defective mutant, while co-transfections in Schneider S2 cells demonstrated dose-dependent trans-activation of -358-AQP5-Luc by Sp1. Activation of -358-AQP5-Luc by GATA-6 was dramatically reduced by Sp1 siRNA, and -358-AQP5-Luc was activated synergistically by GATA-6 and Sp1 in NIH3T3 cells. Furthermore, association between endogenous GATA-6 and Sp1 was demonstrated by co-immunoprecipitation. These results suggest that transcriptional activation of AQP5 by GATA-6 is mediated at least in part through cooperative interactions with Sp1 occurring at the proximal promoter.