A fast and simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in detection and identification of thermophillic lactobacilli cells growing in milk or present in industrial starter cultures. The protocol uses a filtration technique of the samples and epifluorescence microscopy as a detection system, and is completed within 1.5 h. Seven oligonucleotide probes with different ranges of specificity have been tested in in situ hybridization experiments against a number of collection and industrial strains including those of the species Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus pentosus, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc sp. and Streptococcus thermophilus. Certain limitations in the use of DNA probes due to non-specific hybridization were encountered and taken into account. Depending on the probe used, a specific and simultaneous detection of the different species present can be achieved. The equipment used was able to detect between 10(4) and 10(6) cells per mL. The physiological state of a starter cultures of thermophilic lactobacilli can also be evaluated by in situ hybridization. Thus, this fast method can be used for starter cultures and industrial fermentation quality control. This work constitutes the first reported use of PNA molecules for in situ detection and identification of bacteria in milk or in an industrial medium.