During the summer 2005/06, an oceanographic cruise was carried out in the Ross Sea, from Cape Adare, through the Terra Nova Bay polynya to the eastern edge of the Ross Ice Shelf. We analysed microbial activities (prokaryotic carbon production, protease, phosphatase, beta-glucosidase and lipase activity) and bacterial community structure (using Denaturing Gradient Gel Electrophoresis - DGGE) in order to establish if differences in bacterioplankton assemblages and their metabolic requirements occur within the five Ross Sea water masses: Antarctic Surface Waters (AASW), High Salinity Shelf Water (HSSW), Ice Shelf Water (ISW), Antarctic Bottom Water (AABW), Circumpolar Deep Water (CDW). Differences in activities were found between the highly active AASW and all the other water bodies. A Principal Component Analysis highlighted two main gradients: in the Cape Adare area (AASWn, CDW and AABW) higher phosphatase, lipase and glycolytic activities, increasing towards the surface, were identified, whereas in the southern sector of the basin [AASWs and (m)HSSW] higher leucine uptake and polypeptide degradation characterized the second gradient. DGGE fingerprinting showed for the first time that different water masses harboured diverse bacterial communities, highlighting the high specificity of deep water assemblages. Alpha- and Gammaproteobacteria represented the main phylogenetic groupings in all samples and no substantial difference in the phylogenetic composition of assemblages was found between different water masses.