Aims: To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae. Methods and Results: 24-mer oligonucleotide probe (probe D) was designed by comparison of 16S rDNA sequences of 35 species of Enterobacteriaceae, eight species of Vibrionaceae and six species of Pasteurellaceae. The sequence of the probe corresponding to the complementary sequence of a position 1251-1274 of Escherichia coli 16S rRNA was found to be a highly conserved region of 16S rDNA sequence in Enterobacteriaceae different from that of Vibrionaceae and Pasteurellaceae. The fluorescent dye-labelled probe was tested for the specificity by in situ hybridization and epifluorescence microscopy. Seventy-six out of 78 strains belonging to Enterobacteriaceae were visualized in an optimal hybridization condition. No bacterial strains belonging to Vibrionaceae (31 strains) and Gram-positive bacteria (three strains) were visualized. Conclusions: In situ hybridization using probe D allows the detection of bacterial cells belonging to Enterobacteriaceae without false positive reaction. Significance and Impact of the Study: In situ hybridization techniques using the probe D are potential tools for detecting Enterobacteriaceae in food and water samples.