Xylella fastidiosa (X.f.) is a plant pathogen with high levels of genomic similarity to Xanthomonas campestris pv. campestris Mc.c.). It has been shown that X. fastidiosa synthesizes a putative diffusible signal factor (X.f.-DSF) that activates regulation of pathogenicity factor (rpf) genes in a X.c.c. reporter system, which might be involved in the regulation of pathogenesis associated genes as in X.c.c., as well as in quorum-sensing. The nature of the X.f.-DSF is not known, whereas the X.c.c.-DSF has been identified as cis-11-methyl-2-dodecenoic acid. In this work, the chemical nature of a putative X.f.-DSF molecule, able to restore endoglucanase activity in a X.c.c. rpfF mutant, was investigated as if it was a fatty acid derivative. Bioassays with X.c.c. reporter bacterium and X.f. culture extracts, based on endoglucanase restoration activity, were also carried out in order to confirm the DSFs molecules similarities. For this reason, a gas chromatography-mass spectrometry method was developed with standard fatty acids methyl esters mixtures. The retention time, as well as the fragmentation patterns, of each standard was used to identify the DSF molecule synthesized by X.f. in the culture medium. Typical ester fragmentation patterns (the derivatized analyte) were observed, such as: McLafferty rearrangement and migration of the H delta followed by 1,4-hydrogen shift and cleavage of the bond C beta-C gamma, confirming the nature of this molecule. This confirmation was corroborated by the common peaks in both spectra. Besides, the observed retention time reinforces our conclusion since it corresponds to a methyl ester with 15 carbons. Since the X.f.-DSF molecule was tentatively identified as 12-methyl-tetradecanoic acid (by mass spectra library comparison), this standard compound was also analyzed, strongly suggesting that this is the identification of such a molecule. To our knowledge, this is the first time a DSF produced by X.f. has been characterized. Copyright (C) 2007 John Wiley & Sons, Ltd.