Microbial community metabolism and functionality play a key role modulating global biogeochemical processes. However, the metabolic activities and contribution of actively growing prokaryotes to ecosystem energy fluxes remain largely unknown. In this study we describe the temporal and spatial dynamics of active prokaryotes in the different water masses of the open ocean Mediterranean Sea during three seasons using a combination of bromodeoxyuridine labelling and 16S rRNA gene Illumina sequencing.