Microorganisms have been implicated in causing cytoplasmic incompatibility in a variety of insect species, including mosquitoes, fruitflies, beetles and wasps. The effect is typically unidirectional: incompatible crosses produce no progeny or sterile males, whereas the reciprocal crosses produce normal progeny. The parasitic wasp Nasonia vitripennis is one of the few species in which the cytogenetic mechanism of incompatibility is known. In this species the paternal chromosome set forms a tangled mass in a fertilized egg and is eventually lost. Here we report that cytoplasmic microorganisms are associated with complete bidirectional incompatibility between N. vitripennis and a closely related sympatric species, N. giraulti. Microorganisms can be seen in the eggs of both species. Hybrid offspring are normally not produced in crosses between the two species, but do occur after elimination of the microorganisms by antibiotic treatment. A cytogenetic and genetic study shows that bidirectional interspecific incompatibility is due to improper condensation of the paternal chromosomes. Microorganism-mediated reproductive isolation is of interest because it could provide a rapid mode of speciation. The mechanism of incompatibility in Nasonia is also of interest as a potential tool for studying chromosome imprinting and chromosome condensation.