A chemically defined medium, XF-26, supported the in vitro cultivation of two strains of Xylella fastidiosa originally isolated from grapevines with symptoms of Pierce's disease and one strain from almond exhibiting leaf scorch symptoms. Growth of X. fastidiosa in XF-26 was comparable to that in the undefined media CS20 or PD2. Medium XF-26 supported the primary growth of the bacterium isolated from grapevine tissues showing Pierce's disease symptoms as well as CS20 or PD2 did. XF-26 also supported the growth of two other cultivated strains, one originally isolated from periwinkle showing wilt symptoms and the other from oak with leaf scorch symptoms.