Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting. © Springer Science+Business Media, LLC 2012.