Cyprinid herpesvirus 3 (CyHV-3) is a lethal DNA virus that infects common carp and koi. It has caused outbreak of the disease within both aquaculture and natural environmental ecosystems. However, there is not enough understanding of the distribution of CyHV-3 in the natural environments, partly because there is no suitable quantification method. In this study, we tested CyHV-3 extraction methods from sediment and then compared its abundance between sediment and water using real-time PCR. Sediment samples were taken from lake and pond, and total viral DNA was extracted using the viral elution method recommended by the US Environmental Protection Agency (manual method), as well as a commercial DNA extraction kit for soil (commercial kit method) before PCR detection. 7 of 12 (58%) and 5 of 10 (50%) sediment samples showed PCR positive signal for CyHV-3 DNA using the manual method and the commercial kit, respectively, and consistent results were obtained from the samples using the manual method between two independent primer sets. The quantification of CyHV-3 DNA in natural sediment using the manual method and external standard virus revealed that its concentration was 1.2 x 10(4) to 3.3 x 10(5) copies DNA/kg. The concentration in sediments was 46-1238 times higher than that in water from the same location, suggesting that sediment could act as a reservoir for CyHV-3 in natural freshwater environments. This is the first report of the existence of CyHV-3 in the sediment of a natural lake or pond. (C) 2011 Elsevier B.V. All rights reserved.