In this review, we discuss the use of a variety of 3-D models (particularly 3-D skin, lung, breast and endothelial) in radiobiological research and highlight the differences in responses compared to 2-D culturing conditions (monolayers). We review the characteristics of existing 3-D models and aim to point out the substantial advantages 3-D cultures provide for modern radiobiology. In particular, they may facilitate the shift from the classical DNA damage and repair studies mainly carried out in monolayer cultures to the investigation of more generalized responses through pathway analysis and a system biology approach. 3-D models are expected to be very informative for investigations on radiotherapy responses in addressing the low dose risk. However, the 3-D model systems are not as easy to propagate and standardize as monolayer cultures. Therefore, we discuss the problems and limitations of 3-D models and propose ways to overcome some of the problems.