- When cells are transfected with calcium phosphate-precipitated DNA, most of the DNA is internalized through active endocytosis but a small pool is postulated to be internalized by passive diffusion. The relative importance of these two DNA pools in gene transfer and expression has never been examined. We now show that mouse Ltk- fibroblasts internalize greater than 90% of radioactively labeled, calcium phosphate-precipitated DNA by a temperature- and energy-dependent process but a small pool of 2-3% of radioactively labeled DNA may be internalized even at 4 degrees C or under energy depletion conditions. The contribution of these two pools of DNA to gene expression was examined by transfecting Ltk- cells with DNA from a plasmid encoding marker for both transient expression (human growth hormone) and stable integration (neomycin resistance). When cells were depleted of ATP during transfection, transient expression of human growth hormone was reduced to only 2% of control values at day 4 posttransfection. Under the same condition, stable expression of neomycin resistance also was reduced to less than 1% of the control. Therefore, these data showed that only the pool of calcium phosphate-precipitated DNA internalized through an energy-dependent process is ultimately responsible for transient and stable expression of exogenous genes.