The biodiversity of the pelagic bacterioplankton community of a maritime Antarctic freshwater lake was examined by cultivation-dependent and cultivation-independent techniques to determine predominant bacterioplankton populations present. The culture-dependent techniques used were direct culture and observation, polymerase chain reaction amplification of 16S rRNA gene fragments, restriction fragment length polymorphism (RFLP) analysis followed by selective sequencing and fatty acid methyl ester analysis. The culture-independent techniques used were 16S ribosomal DNA gene cloning, RFLP analysis and sequencing, in situ hybridisation with group-specific, fluorescently labelled oligonucleotide probes and cloning and sequencing of dominant denaturing gradient gel electrophoresis products. Significant differences occurred between the results obtained with each method. However, sufficient overlap existed between the different methods to identify potentially significant groups. At least six different bacterial divisions including 24 genera were identified using culture-dependent techniques, and eight different bacterial divisions, including 23 genera, were identified using culture-independent techniques. Only five genera, Corynebacterium, Cytophaga, Flavobacterium, Janthinobacterium and Pseudomonas, could be identified using both sets of techniques, which represented four different bacterial divisions. Significantly for Antarctic freshwater lakes, pigment production is found within members of each of these genera. This work illustrates the importance of a comprehensive polyphasic approach in the analysis of lake bacterioplankton, and supports the ecological relevance of results obtained in earlier entirely culture-based studies.