Bacteria of the genus Wolbachia (Rickettsiae) are endosymbionts found in many species of invertebrates. The traditional division of Wolbachia lines into four supergroups has recently been challenged with the finding of a fifth supergroup. Until now, this E supergroup exists of a single host species of the order Collembola (springtails) and is hypothesized to form a 'missing link' between Wolbachia lines in higher arthropods and nematodes. We have sequenced the 16S rDNA, the ftsZ and the wsp genes of a Wolbachia lineage present in the sexually reproducing collembolan Orchesella cincta. We tested the hypothesis on the origin of Wolbachia in Collembola by adding this new Wolbachia lineage and a lineage found in the collembolan Mesophorura macrochaeta to the Wolbachia phylogenetic tree. The addition of these two species clearly shows that Wolbachia is not monophyletic in Collembola. We conclude that the origin of Wolbachia in Collembola is a combination of vertical and horizontal transfer between coexisting species. © 2004 Elsevier GmbH. All rights reserved.