To decode the function and molecular recognition of several recently discovered cytosine derivatives in the human genome – 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine – a detailed understanding of their effects on the structural, chemical, and biophysical properties of DNA is essential. Here, we review recent literature in this area, with particular emphasis on features that have been proposed to enable the specific recognition of modified cytosine bases by DNA-binding proteins. These include electronic factors, modulation of base-pair stability, flexibility, and radical changes (...) in duplex conformation. We explore these proposals and assess whether or not they are supported by current biophysical data. This analysis is focused primarily on the properties of epigenetically modified DNA itself, which provides a basis for discussion of the mechanisms of recognition by different proteins. 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine have recently been discovered in mammalian DNA and appear to have essential regulatory roles. Here, we summarize recent investigations into their effects on the structural, chemical, and biophysical properties of DNA, an understanding of which is essential to decoding their behavior in the genome. (shrink)