Formaldehyde has been used extensively for chemically modifying proteins and for inactivating toxins and viruses, although the precise mode of its action is little understood. In the case of tobacco mosaic virus, however, Fraenkel-Conrat (1) and Staehelin (2) have shown that inactivation of the virus is due to interaction of formaldehyde with the amino groups of adenine, cytosine, and guanine in the ribonucleic acid component. Fraenkel-Conrat has suggested that the products of formylation are Schiff bases, of the type R N = CH,. Recently, Hoard has shown by means of a form01 titration that the amino group of cytidylic acid combines with one molecule of formaldehyde in the limit of high formaldehyde concentration, which is consistent with Fraenkel-Conrat’s suggestion (3). Fraenkel-Conrat (I), Staehelin (2), and Zamenhof et al. (4) have all reported that native DNA from several sources does not react with formaldehyde under the same conditions used for the reaction with RNA. The conclusion drawn was that amino groups involved in hydrogen bonds do not react with formaldehyde. If this is true, then the extent of reaction of a particular nucleic acid with formaldehyde, for a given set of experimental conditions (pH, temperature, ionic strength, and formaldehyde concentration) should reflect quantitatively the fraction of amino groups involved in hydrogen bonds. It has recently been demonstrated that the temperature dependence of the rate of reaction with formaldehyde is considerably greater for TMVl-RN,4 than for a mixture of the constituent mononucleotides. Qualitatively this has been explained on the basis of thermal rupture of hydrogen bonds in the intact nucleic acid at elevated temperatures, which make the amino groups more accessible to the reagent (5). In view of these considerations, the present work was undertaken to explore the possibility of using the reaction with formaldehyde as a quantitative measure of the fraction of hydrogen bonded amino groups in nucleic acids. First, we consider in detail the effect of temperature and ionic strength on the rate of reaction of formaldehyde with monoand polynucleotides. This leads to a hypothesis concerning the mode of action of formaldehyde which is tested and verified in the following section. Finally, this information is used to establish conditions for determining the helical content of TMV-RNA. The results of such a determination are compared with other estimates.