The bacterioplankton community structure in Moss Lake, a maritime Antarctic oligotrophic lake, was determined with vertical depth in the water column, during the ice-free period on Signy Island in the South Orkney Islands. Bacterioplankton community structure was determined using a combination of direct counting of 4′,6-diamidino-2-phenylindole (DAPI) stained cells, PCR amplification of 16S rRNA gene fragments, denaturing gradient gel electrophoresis (DGGE) and in situ hybridization with group-specific, fluorescently labeled oligonucleotide probes. Using PCR amplification of 16S rRNA gene fragments and DGGE, the bacterioplankton community composition was shown to be constant with vertical depth in the water column. Specific bacterioplankton species identified through cloning and sequencing the DGGE products obtained were Flavobacterium xinjiangensis (a Flavobacterium), Leptothrix discophora (a beta-Proteobacterium), and a number of uncultured groups: two beta-Proteobacteria, an unclassified Proteobacterium, three sequences from Actinobacteria, and a Cyanobacterium. Fluorescence in situ hybridization (FISH), however, demonstrated that there were minor but significant fluctuations in different groups of bacteria with vertical depth in the water column. It showed that the beta-Proteobacteria accounted for between 26.4 and 71.5%, the alpha-Proteobacteria 2.3–10.6%, the gamma-Proteobacteria 0–29.6%, and the Cytophaga–Flavobacterium group 1.8–23.5% of cells hybridizing to a universal probe. This study reports the first description of the community structure of an oligotrophic Antarctic freshwater lake as determined by PCR-dependent and PCR-independent molecular techniques. It also suggests that the bacterioplankton community of Moss Lake contains classes of bacteria known to be important in freshwater systems elsewhere in the world.