Infection of human endothelial cells with first-generation E1(-)E4(+) adenovirus (Ad) vectors leads to prolonged cell survival and changes in the cell phenotype to a more quiescent stage. Based on the concept that the CXCR4, the receptor for the endothelial chemoattractant stromal-derived factor-&alpha (SDF-alpha), is constitutively expressed by quiescent, resting endothelial cells, the present study analyzes the effect of Ad vector infection on CXCR4 expression and SDF-alpha responses of human umbilical vein endothelial cells (HUVEC). CXCR4 transcripts were markedly downregulated in E1(-)E4(+) Ad-infected cells 48 h following infection, but not in uninfected control cells or when the cells were infected with an E1(-)E4(-) Ad vector. Analysis of surface CXCR4 expression by flow cytometry demonstrated marked reduction of the CXCR4 receptor on cells infected with E1(-)E4(+) Ad compared to uninfected control cells or E1(-)E4(-) Ad-infected cells. Infection of other cell types which express CXCR4, such as dendritic cells and myeloma cells, did not exhibit CXCR4 receptor downregulation following infection with E1(-)E4(+) Ad. Consistent with the observed downregulation of CXCR4 mRNA and surface protein, infection of the endothelial cells with an E1(-)E4(+) Ad rendered the cells unresponsive to the chemoattractant SDF-alpha compared to naive or E1(-)E4(-) Ad-infected cells. Together, the data suggest that first-generation Ad vectors, likely the E4 region, modify the ability of endothelial cells to respond to at least one important chemoattractant.