Xylella fastidiosa is a bacterial plant pathogen that causes many plant diseases, including Pierce’s disease of grapevines. Sharpshooter leafhopper (Hemiptera: Cicadellidae: Cicadellinae) vectors transmit this bacterium to plants. Although the basic mechanism of pathogen transmission is not completely understood, previous studies implicated the foregut of infected insects as the source of bacterial inoculum because infective nymphs lose transmissibility after molting and no latent period is required for transmission. Scanning electron microscopy documented the spatial distribution of X. fastidiosa in the foreguts of the sharpshooter Graphocephala atropunctata (Signoret) after short (1-d) and long (4-d) acquisition access periods on infected plants and various inoculation access periods on grape test plants. After 1-d acquisition and 1-d inoculation periods, cells had only colonized portions of the precibarium between the stylets and the cibarial pump, often embedded in a matrix-like material. After long (15-d) periods after acquisition, we found bacterial cells attached polarly to the insect’s cuticle in a regular pattern throughout the precibarium but absent from specific locations on both pharynges. Insects sampled for microscopy 2 wk after long acquisition access periods and that contained bacteria in their mouthparts transmitted X. fastidiosa to healthy grapevines independently of the length of the acquisition access period. In contrast, individuals free of bacteria on the precibarium did not transmit the pathogen. After successive short acquisition and inoculation periods, 5 of 12 X. fastidiosa-positive insects did not transmit to plants. We also did transmission experiments with the sharpshooter Homalodisca coagulata; however, only one of 30 individuals had X. fastidiosa attached to its precibarium, and none transmitted it to plants. Our results suggest that sharpshooters introduce into plants X. fastidiosa cells that detach from the precibarial canal during feeding.