A new in situ hybridization technique was developed for identification of Bartonella henselae cells in cell suspension or in tissue sections. Use of highly specific probes labeled with either fluorescein or digoxigenin allows discrimination between B. henselae and closely related B. quintana cells. No cross-hybridization with other Bartonella or non-Bartonella species was observed. Besides its specificity it showed higher sensitivity as compared to PCR based detection methods. Moreover, its application allows direct observation of B. henselae in infected tissues.