The taxonomic position of Photorhabdus strains was examined through the results of DNA relatedness (S1 nuclease method) studies associated with the determination of delta Tm, 16S rRNA phylogenetic inferences and phenotypic characterization, including morphological, auxanographic, biochemical and physiological properties. Three genomic species were delineated on a consensus assessment. One of these species corresponded to Photorhabdus luminescens, since strains were at least 50% related to the type strain of this species with delta Tm less than 7 degrees C. The two other species were novel genomic species II and III, which were less than 40% related to each other with delta Tm higher than 9 degrees C. A comparison of the complete 16S rDNA sequences of several representatives of genomic species II and genomic species III revealed that each of them formed a stable lineage independent of the cluster generated by P. luminescens strains. The genomic species differed in their maximum temperatures for growth. A correlation with the ecological origin of the bacterial samples was noticed. The heat-tolerant group I (maximum growth temperature 35-39 degrees C) corresponded to the symbionts of Heterorhabditis bacteriophora groups Brecon and HP88 and Heterorhabditis indica, nematodes living in warm and tropical countries, respectively. Group II (maximum growth temperature 33-35 degrees C) encompassed symbionts from Heterorhabditis megidis, Heterorhabditis zealandica and group NC1 of H. bacteriophora, nematodes isolated in temperate climates. Group III were bacteria isolated from human specimens. Two new species, Photorhabdus temperata sp. nov. (type strain CIP 105563T) and Photorhabdus asymbiotica sp. nov. (type strain ATCC 43950T), are proposed for genomic species II and III, respectively. Species I and II can be separated into sub-groups on the basis of high DNA-DNA relatedness (more than 80% DNA binding with delta Tm < 1.5 degrees C), 16S rDNA branching and phenotypic characters. Therefore, we propose that the two species P. luminescens and P. temperata should be subdivided into subspecies as follows: P. luminescens subsp. luminescens subsp. nov. (type strain ATCC 29999T), P. luminescens subsp. akhurstii subsp. nov. (type strain CIP 105564T), P. luminescens subsp. laumondii subsp. nov. (type strain CIP 105565T) and P. temperata subsp. temperata subsp. nov.