In situ hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect microorganisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic Candida species, we have designed an oligonucleotide probe which binds to the 18S rRNA of C. albicans and C. tropicalis, the two most important pathogenic Candida species, and differentiates them from other clinically relevant species. After establishing suitable hybridization conditions, we confirmed the specificity of our probe O20 in RNA dot blot hybridizations with a series of reference strains and clinical isolates of medically important Candida species. All C. albicans and C. tropicalis strains hybridized with the probe, whereas all strains of C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. kefyr and C. lusitaniae did not. When we used the fluorescently labelled probe O20 to specifically detect single cells of the two target species by in situ hybridization, both C. albicans and C. tropicalis reacted strongly with the probe and could be clearly differentiated from C. krusei and C. parapsilosis, although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity was also seen when mixed suspensions of C. albicans and C. parapsilosis were hybridized with the probe. After infection of a human endothelial cell line with C. albicans and C. krusei, C. albicans cells adhering to the endothelial cells were easily distinguishable from the C. krusei cells by fluorescent in situ hybridization with probe O20. In addition, germ tubes and hyphae of C. albicans were also efficiently labelled. The application of fluorescently labelled rRNA-targeted oligonucleotide probes therefore appears to be a valuable tool for the specific detection and identification of different members of the genus Candida, which does not require any cultivation.