Microscopic localization of endosymbiotic bacteria in three species of mealybug (Pseudococcus longispinus, the long-tailed mealybug; Pseudococcus calceolariae, the citrophilus mealybug; and Pseudococcus viburni, the obscure mealybug) showed these organisms were confined to bacteriocyte cells within a bacteriome centrally located within the hemocoel. Two species of bacteria were present, with the secondary endosymbiont, in all cases, living within the primary endosymbiont. DNA from the dissected bacteriomes of all three species of mealybug was extracted for analysis. Sequence data from selected 16S rRNA genes confirmed identification of the primary endosymbiont as "Candidatus Tremblaya princeps," a betaproteobacterium, and the secondary endosymbionts as gammaproteobacteria closely related to Sodalis glossinidius. A single 16S rRNA sequence of the primary endosymbiont was found in all individuals of each mealybug species. In contrast, the presence of multiple divergent strains of secondary endosymbionts in each individual mealybug suggests different evolutionary and transmission histories of the two endosymbionts. Mealybugs are known vectors of the plant pathogen Grapevine leafroll-associated virus 3. To examine the possible role of either endosymbiont in virus transmission, an extension of the model for interaction of proteins with bacterial chaperonins, i.e., GroEL protein homologs, based on mobile-loop amino acid sequences of their GroES homologs, was developed and used for analyses of viral coat protein interactions. The data from this model are consistent with a role for the primary endosymbiont in mealybug transmission of Grapevine leafroll-associated virus 3.