16S rRNA gene libraries were prepared by polymerase chain reaction amplification and cloning from soil samples taken periodically from a field with genetically modified plants. Sequence analyses of the cloned rDNAs indicated that 140 of them clustered apart from known bacterial phyla. Based on 31 full sequences a new phylum could be defined. It includes Holophaga foetida, ‘Geothrix fermentans’ and Acidobacterium capsulatum as the only cultured species so far. Therefore, this line of descent was named the Holophaga/Acidobacterium phylum. About 50 published partial sequences of cloned rDNAs retrieved from soil, freshwater sediments or activated sludge from different continents indicate the occurrence of further representatives of this phylum. Two specific hybridization probes were constructed for members of one of four subclusters. A careful data analysis revealed the importance and problems of identifying and dealing with artefacts such as chimeric structure when defining new phylogenetic groups based mainly upon cloned amplified rDNAs. For the first time, the presence of bacterial cells representing this group could be shown in soil, sediment, activated sludge and lake snow by in situ hybridization.