The fluorescent amplified fragment length polymorphism (FAFLP) groups A5 (21 isolates), A8 (6 isolates) and A23 (3 isolates) distinguished in an earlier paper (Thompson et al., Syst Appl Microbiol 24, 520-538, 2001) were examined in more depth. These three groups were phylogenetically related to Vibrio tubiashii, but DNA-DNA hybridization experiments proved that the three AFLP groups are in fact novel species. Chemotaxonomic and phenotypic analyses further revealed several differences among the 30 isolates and known Vibrio species. It is proposed to accommodate these isolates in three novel species, namely Vibrio neptunius (type strain LMG 20536T; EMBL accession no. AJ316171; G +C content of the type strain 46.0 mol%), Vibrio brasiliensis (type strain LMG 20546T; EMBL accession no. AJ316172; G + C content of the type strain 45.9 mol%) and Vibrio xuii (type strain LMG 21346T; EMBL accession no. AJ316181; G +C content of the type strain 46.6 mol%). These species can be differentiated on the basis of phenotypic features, including fatty acid composition (particularly 14:0 iso, 14:0 iso 3-OH, 16:0 iso, 16:0, 17:0 and 17:1 omega8c), enzyme activities and utilization and fermentation of various carbon sources.