A novel method was developed for the quantification of dissolved deoxyribonucleic acid (D-DNA) in seawater. This method includes addition of tetrasodium ethylenediamine tetraacetic acid (tetrasodium EDTA) to 0.22 μm-filtered seawater, concentration of > 10 kDa material in the filtrate with a Centricon centrifugal concentration unit, and quantification of the concentrated D-DNA with the fluorescent double-stranded DNA stain SYBR Green I. This method requires less than 15 mL of seawater per sample even in oligotrophic environments, and samples can be analyzed in approximately 3 h. The recovery of D-DNA with this method is 75% to 85% and can be determined for each sample by measuring recovery of 35S-labeled DNA added at trace amounts. This method can be used to quantify D-DNA concentrations as low as 0.01 ng mL -1 with high precision (standard deviation < 5% of the mean). Deoxyribonuclease (DNase) treatment of samples and virus enumeration can be used in conjunction with this method to determine the three major components of D-DNA: free or enzymatically hydrolyzable DNA (ehD-DNA), DNA within viruses, and uncharacterized bound DNA. © 2004, by the American Society of Limnology and oceanography, Inc.