The taxonomic status of the genus Acinetobacter is currently confused and the role of these organisms in activated sludge is poorly understood. Currently unidentified isolates of Acinetobacter from activated sludge were fingerprinted by making use of polymorphisms in their 16S-23S rDNA spacer region. The PCR amplified 16S-23S rDNA spacer region was digested with five different restriction enzymes to further differentiate between the isolates. The resulting band patterns were very diverse and the data suggests that the activated sludge isolates are different to the known genomic species of Acinetobacter which are predominantly clinical isolates. The results of this study imply the existence of yet unrecognised species of Acinetobacter in activated sludge.