Aims: To develop a species-specific probe (VSV3) for the detection of Vibrio scophthalmi in fish intestine and to apply this probe to study the host specificity of V. scophthalmi . Methods and Results: A specific probe (VSV3) based on the variable region V3 of the 16S rRNA gene (rDNA) was designed. Its specificity was tested by DNA-DNA hybridization and by colony hybridization. No cross-hybridization was found. The sensitivity of the probe was tested both by DNA-DNA hybridization and by colony hybridization. The detection limit of V. scophthalmi 16S rDNA was 150 pg or 10 cfu. Vibrio scophthalmi cells were detected in experimental samples constituted by mixed cultures when present in proportions of 1 : 10 and 1 : 100. The VSV3 probe also proved to be reliable for the detection of V. scophthalmi in samples of fish intestine. Conclusions: The VSV3 probe can be used for the detection of V. scophthalmi in colony hybridization or DNA-DNA hybridization of amplified 16S rDNA. Preliminary results indicate that V. scophthalmi may present certain host specificity for turbot. Significance and Impact of the Study: The VSV3 probe provides a useful tool for ecological studies.