Biomarkers (respiratory quinones and cellular fatty acids) and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes were used to characterize the microbial community structure of lab-scale enhanced biological phosphate-removal (EBPR) systems in response to altering sludge phosphorus (P) content. All the data suggest that the microbial community structures of sludge samples with a P content between 8 and 123 % (sludge dry weight) (i.e. good EBPR activity) were very similar, but differed from those with 2 % P content (i.e. no EBPR activity). For all samples analysed, ubiquinones Q-8 and Q-10, menaquinone MK-8(H 4 ), and fatty acids C 16 :0 , C 16 :1 ω9c and C 18 :1 ω11c were the major components. The dominance of Q-8, Q-10 and MK-8 (H 4 ) suggested that large numbers of organisms belonging to the β and α subclasses of the Proteobacteria and the Actinobacteria from the high GMC Gram-positive bacteria, respectively, were present. DGGE analysis revealed at least 7-9 predominant DNA bands and numerous other fragments in each sample. Five major DGGE fragments from each of the 2 % and 12 % Pcontaining sludge samples, respectively, were successfully isolated and sequenced. Phylogenetic analysis of the sequences indicated that both 2 % and 12 % P-containing sludge samples shared three common phylotypes that were separately affiliated with a novel bacterial group from the γ subclass of the Proteobacteria, two MK-8(H 4 )-containing actinobacteria previously isolated from the 2 % P-containing sludge, and a Caulobacter spp. in the α subclass of the Proteobacteria. The phylogenetic analysis also revealed phylotypes unique to both sludge samples. Changes in sludge P content therefore had an effect on the composition and abundance of the predominant microbial populations, though specific phylotypes could not be unequivocally associated with EBPR.