Nutrient agar (NA) was compared with CS20 and PD2 media for isolation of Xylella fastidiosa, the causal bacterium of Pierce's disease of grapevines. Branches showing Pierce's disease symptoms were collected from muscadine grapes (Vitis rotundifolia), American bunch grapes (Vitis labrusca), and French hybrids (Vitis vinifera x V. labrusca). Surface-sterilized petioles were comminuted in PD2 broth medium, and the resulting suspension was streaked onto all three media. NA as well as CS20 and PD2 media supported the primary isolation of X. fastidiosa. Strains isolated in NA medium were injected into healthy grapes and they induced typical symptoms as did those isolated in CS20 and PD2 media. The bacterium was reisolated from the inoculated plants, fulfilling Koch's postulates.