Two grape strains (FC and C) of the causal organism of Pierce’s disease (PD) of grapevine, Xylella fastidiosa, were isolated from infected grape petioles onto PD3 medium, nutrient agar (NA), and NA amended with various concentrations of sucrose (NAS). All media supported growth of both strains, however, the C strain grew more slowly than the FC strain. Four additional strains from grape grew as single colonies on each of the media. The FC and C strains were subcultured weekly on PD3 and nutrient agar-2.0% sucrose media for 12 mo. The strains remained pathogenic to the grapevine cultivar French Colombard but became less aggressive after 8 mo of subculturing. Colonies of X. fastidiosa were reisolated from leaves with symptoms of Pierce’s disease.