Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue samples mounted on glass slides. Two different methods are presented: one is illustrated with the use of peptide nucleic acid (PNA) that is carried out directly on glass slides (Method I), whereas the other is exemplified by using a DNA probe in a Shandon rack (Method II). In the two methods, both PNA and DNA probes can be used.