Aromatic NH2 groups are essential as hydrogen-bond donors in secondary structures of DNA and RNA. Although rapid rotations of NH2 groups of adenine and guanine bases were previously characterized, there has been a lack of quantitative information about slow rotations of cytosine NH2 groups in Watson-Crick base pairs. In this study, using an NMR method we had recently developed, we determined the kinetic rate constants and energy barriers for cytosine NH2 rotations in a 15-base-pair DNA duplex. Our data show that the rotational dynamics of cytosine NH2 groups depend on local environments. Qualitative correlation between the ranges of 15N chemical shifts and rotational time scales for various NH2 groups of nucleic acids and proteins illuminates a relationship between the partial double-bond character of the C-N bond and the time scale for NH2 rotations.