Aims: To design a rapid specific method for enumeration of viable Listeria spp. using the fluorescence in situ hybridization with filter cultivation (FISHFC) method. Methods and Results: The probe, Lis-1400, was designed from the 23S rRNA region of the Listeria genome, and labelled with 5'-carboxy-tetramethyl-rhodamine-N- hydroxy-succinimide-ester. Fluorescence was observed for all Listeria species but not for any organisms from the other genera, suggesting Lis-1400 is highly specific for Listeria spp. For purposes of filter cultivation prior to hybridization, hydrophilic polypropylene membrane filters gave better contrast between fluorescing colonies and background fluorescence. This was because of a high S/N ratio (fluorescence intensity of each microcolony/fluorescence intensity of background noise) after FISH treatment. Results were achievable in 14 h using Lis-1400-aided FISHFC as compared with 4-7 days required for confirmation of Listeria spp. by conventional plate count methods. Moreover, viable Listeria counts in selected food samples showed no significant differences between Lis-1400-aided FISHFC and conventional methods. Conclusions: The Lis-1400-aided FISHFC method is more efficient than conventional methods for enumeration of viable Listeria spp. in food samples. Significance and Impact of the Study: For enumeration of Listeria spp., Lis-1400-aided FISHFC method is equally accurate yet faster than conventional plate count methods, and can be valuable in the control of listeriosis.