the murine urothelial cell line, mb49 was transfected with the reporter gene pcmvlacz using a number of commercial transfection agents. the transfection efficiency of these agents, as determined by beta-galactosidase activity, is in the order of dotap>superfect>Fugene. The addition of methyl-beta-cyclodextrin solubilized cholesterol (MBC) to Dotap and Superfect further improved their transfection efficiency by 3.8-fold and 2.6-fold, respectively. beta-Galactosidase activity was detectable within 1 h of transfection and peaked at 48 h. Nuclear and cytoplasmic separation showed that with Dotap + methyl-beta-cyclodextrin solubilized cholesterol (DMBC), the DNA plasmid complex was found in both the nucleus and the cytoplasm. In vivo, murine bladders were transfected with an intravesical instillation of DMBC + DNA for 2 h. Two days later the bladder, lungs, liver, spleen and heart were assayed for the presence of the beta-galactosidase gene by staining and PCR. Expression of the gene was confined to the bladder. Both in vitro and in vivo expression was observed after as little as a 15 min exposure to DMBC:DNA. Expression of the marker gene was present up to 30 days after transfection in vivo. From our data it appears that DMBC is the best nonviral agent for the transfection of urothelial cells in vitro and in vivo.