In living organisms, DNA is generally confined into very small volumes. In most viruses, positively charged multivalent ions assist the condensation of DNA into tightly packed toroidal structures. Interestingly, such cations can also induce the spontaneous formation of DNA toroids in vitro. To resolve the condensation dynamics and stability of DNA toroids, we use a combination of optical tweezers and fluorescence imaging to visualize in real-time spermine-induced (de)condensation in single DNA molecules. By actively controlling the DNA extension, we are able to follow (de)condensation under tension with high temporal and spatial resolution. We show that both processes occur in a quantized manner, caused by individual DNA loops added onto or removed from a toroidal condensate that is much smaller than previously observed in similar experiments. Finally, we present an analytical model that qualitatively captures the experimentally observed features, including an apparent force plateau. © 2010 by the Biophysical Society.