Using flow cytometry, single cell sorting, confocal microscopy and fluorescent plasmids, a thorough study of DNA uptake, DNA fate and DNA expression in mammalian cells transfected with the widely used calcium-phosphate precipitation method was executed. We show for the first time that up to 100,000 plasmid molecules can be delivered into individual cells, but also that DNA transfer into cells is a dynamic process that follows a defined kinetics of uptake and intracellular processing. Analyses by flow cytometry and confocal microscopy have also supported results suggesting endocytosis during Ca-Pi transfection. We also demonstrate that expression-enhancing treatment with glycerol during transfection did not result in increased DNA uptake. While cells with maximal DNA load appear to express the highest level of the transgene, these cells are negatively impacted in terms of growth and survival.