Restriction maps of PCR-amplified 16s rRNA genes (rrs for ribosomal rRNA small subunit) were prepared and compared for 43 Xanthomonas and Stenotrophomonas strains belonging to representative DNA-DNA similarity groups. Nineteen restriction fragment length polymorphisms (RFLPs) were distinguished, forming two main clusters: one contained all Xanthomonas species, the other contained strains of Stenotrophomonas spp. (formerly named X. maltophilia). The genera Xanthomonas and Stenotrophomonas appeared to form a coherent group excluding Xylella fastidiosa. The rrs RFLP permitted to differentiate X. fragariae, X. oryzae, X. axonopodis, X. populi pv. populi from most pathovars of X. campestris. The latter species had very closely related patterns, contrary to X. albilineans, X. campestris pv. hyacinthi and pv. graminis, and a X. campestris strain (isolated from Saccharum officinarum, Guadeloupe) whic formed a more distinct subcluster. All genospecies of Stenotrophomonas had different rrs patterns, and appeared to form a less coherent taxon. Relationships between the percentage of DNA-DNA hybridization and the rate of nucleotide substitution in rrs confirmed the clear delineation of the two genera.