Clue cells are epithelial cells covered by adherent gram-negative rods, observed in vaginal smears from women with bacterial vaginosis. Immunofluorescence studies were used to identify the gram-negative bacteria adhering to clue cells. Specific antisera to four common gram-negative vaginal bacteria (Gardnerella, Bacteroides, Fusobacterium, and Mobiluncus) were prepared by long-term, multiple, small-inoculum immunization of rabbits. Cross-reactivity with heterologous common vaginal bacteria was removed by absorption against whole cells of heterologous bacteria and by serial dilution. Gardnerella vaginalis was most often observed adhering to the surface of clue cells and was detected on the surface of exfoliated vaginal epithelial cells significantly more frequently and in higher numbers than were Mobiluncus, Bacteroides, and Fusobacterium, suggesting that this species of gram-negative bacteria is responsible for clue cell formation.