The transfer of nucleic acids into living cells, i.e. transfection, is a major technique in current molecular biology and medicine. As nucleic acids alone are not able to penetrate the cell membrane, an efficient carrier is needed. Calcium phosphate nanoparticles can serve as carrier due to their biocompatibility, biodegradability and high affinity to nucleic acids like DNA or RNA. Their application was extended here from two-dimensional (2D) to three-dimensional (3D) cell culture models, including co-cultures. Compared to 2D monolayer cell cultures, a 3D culture system represents a more realistic spatial, biochemical and cellular environment. The uptake of fluorescent calcium phosphate nanoparticles (diameter 40-70 nm; cationic) was studied in 2D and 3D cell culture models by confocal laser scanning microscopy. The transfection of eGFP by calcium phosphate nanoparticles was compared in 2D and 3D cell culture, including co-cultures of green fluorescing HeLa-eGFP cells and MG-63 cells in 2D and in 3D models with the red fluorescent protein mCherry. This permitted a cell-specific assessment of the local transfection efficiency. In general, the penetration of nanoparticles into the spheroids was significantly higher than that of a model oligonucleotide carried by Lipofectamine. The transfection efficiency was comparable in 3D cell cultures with 2D cell cultures, but it occurred preferentially at the surface of the spheroids, following the uptake pathway of the nanoparticles. STATEMENT OF SIGNIFICANCE: Three-dimensional cell culture models can serve as a bridge between the in-vitro cell cultures and the in-vivo situation, especially when mass transfer effects have to be considered. This is the case for nanoparticles where the incubation effect in a two-dimensional cell culture strongly differs from a three-dimensional cell culture or a living tissue. We have compared the uptake of nanoparticles and a subsequent transfection of fluorescent proteins in two-dimensional and three-dimensional cell culture models. An elegant model to investigate the transfection in co-cultures was developed using HeLa-eGFP cells (green fluorescent) together with MG-63 cells (non-fluorescent) that were transfected with the red-fluorescing protein mCherry. Thereby, the transfection of both cell types in the co-culture was easily distinguished.