The luciferase reporter gene incorporated into adenoviral vectors is very useful for monitoring viral transduction of different cell types or for comparing the transduction efficiency of different viral constructs of one cell type. Luciferase protein expression can be detected and quantified with very high sensitivity from whole cells or organ extracts. However, its disadvantages become obvious when aiming at evaluation of transduction events at the single cell level. The results obtained from whole cell extracts cannot be directly correlated to single cell events. In this paper direct cellular luciferase imaging using cell permeable luciferin substrates is applied for comparative analysis of cellular transduction events by two adenoviral vectors with different cell binding properties. Using digital imaging microscopy we show a more than ten-fold increase in transduction efficiency by Ad5LucRGD vectors versus AdCMVLuc vectors on human A549 cells.