A cytoplasmically inherited microorganism associated with male killing in the two-spot ladybird beetle, Adalia bipunctata, is shown to be closely related to bacteria in the genus Rickettsia. Sequencing of a PCR-amplified product of the 16S genes coding for rRNA (16S rDNA) shows the organism associated with male killing in ladybirds to share a common ancestry with the Rickettsias relative to other genera (e.g., Anaplasma, Ehrlichia, and Cowdria). The rickettsial 16S rDNA product is found in four strains of ladybird beetle showing male embryo lethality and is absent from two uninfected strains and an antibiotic-cured strain. In addition, a revertant strain that had naturally lost the male-killing trait failed to amplify the rickettsial 16S rDNA product. Use of PCR primers for a 17-kDa protein antigen which is found only in rickettsias also resulted in an amplified product from infected strains. Uninfected, cured, and revertant strains and insect species infected with related bacteria (cytoplasmic-incompatibility bacteria from Nasonia wasps) failed to amplify the product. Discovery of a close relative of rickettsias associated with sex ratio distortion in insects has implications for the evolution and population dynamics of this bacterial genus.