Molecular and culture-based methods were used to investigate the microbial diversity in produced water obtained from the high-temperature Troll oil formation in the North Sea. 16S rRNA gene libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide primer sets. Sequence analysis of 88 clones in the bacterial library indicated that they originated from members of Firmicutes (48 sequences), Bacteroidetes (17 sequences), delta-Proteobacteria (15 sequences), Spirochaetes (5 sequences), Thermotogales (2 sequences) and gamma-Proteobacteria (1 sequence). Twenty-two sequences in the archaeal library were close relatives to members of the genera Methanococcus (18 sequences), Methanolobus (3 sequences) and Thermococcus (1 sequence). Most of the bacterial sequences shared less than 95% identity with their closest match in GenBank, indicating that the produced water harbours a unique community of novel bacterial species or genera. Members of the thermophilic genera Thermosipho, Thermotoga, Anaerophaga and Thermovirga were isolated. The Troll formations are not injected with sea water. Thus, dramatic changes of the in situ conditions have been avoided, and a common source of continuous contamination from injection water can be excluded. However, the majority of the organisms detected in the gene libraries were most closely related to cultivated organisms with optimum temperatures for growth well below the in situ reservoir temperature (70 degrees C), indicating that produced water from the Troll platform harbours a substantial amount of non-indigenous organisms. This was confirmed by the isolation of a number of mesophilic and moderately thermophilic organisms that were unable to grow at reservoir temperature.