Shrimp farming is a small but growing industry in New Caledonia. Since 1993, "Syndrome 93" has been affecting New Caledonian shrimp farming industry every cold season, causing severe epizootic mortalities in grow-out ponds and significant losses. Highly pathogenic strains of Vibrio penaeicida are considered the etiological agent of the disease in Litopenaeus stylirostris. On one hand, studies demonstrated that healthy shrimp may carry V penaeicida for weeks with a high overall prevalence, regardless of any seasonal pattern or temperature conditions. On the other hand, larvae are free of V penaeicida and are also resistant to experimental infection. V penaeicida is frequently detected in incoming water pumped from the bays, which was shown, by a molecular typing study, to be the infectious source. This particular epidemiological pattern highlights the major role of the factors that trigger and aggravate the disease in grow-out ponds, where shrimp populations carry the pathogen all year round. In order to gain a better understanding of "Syndrome 93" epidemiology, quantification of V penaeicida both in shrimp and the shrimp farm ecosystem is necessary. This article describes the steps in the successful development of a real-time PCR quantification assay of V penaeicida in shrimp haemolymph, seawater (from ponds or bays) and sediment pore water, including the choice of an accurate extraction technique. The entire detection method; including sample processing, DNA extraction and real-time PCR amplification, can be completed within 4 h. (c) 2006 Elsevier B.V All rights reserved.