We have examined transfection conditions of chinese hamster ovary cells using FuGENE6 and immortalized gonadotrope cell line LbetaT2 cells using Lipofectamin 2000 and to obtain reproducible and reliable transfection. The experiments were performed with fluorescent protein expression vectors, pEYFP-C1 and pECFP-C1, or secreted-type alkaline phosphatase vector, pSEAP2, as reporter genes. The number of cells that received reporter plasmid increased in proportion to the amount of DNA and reached a plateau at a large amount. Co-transfection using two fluorescence vectors with a small amount of DNA demonstrated that every transfected cell received both vectors without discrimination. The results further indicate that there is a hierarchy of DNA receptiveness among competent cells. Simultaneously, we observed that a reliable transfection took place at the high dose of DNA. That is, the addition of carrier DNA makes possible a reliable delivery of a small amount of DNA of interest to the competent cells. Similar results were also obtained by pSEAP2 vector. Co-transfection of pEYFP-C1 and pECFP-C1 with various ratios at adequate amounts demonstrated that the fluorescence intensities by each vector are proportional to each amount of vector used with comparable efficiency. In addition, we observed that the variation of the assay using fluorescent vectors or secreted alkaline phosphatase vectors were small enough within the +/- 25% (SD, n = 4), showing that the internal marker often used to normalize the data is not essential, since the vectors used allow us to exclude cell-harvest and cell-lysis. Thus, the present study demonstrates that the addition of carrier DNA during transfection provides reproducible and reliable results.