The gram-negative, anaerobic bacterium Prevotella intermedia plays an important role in the progression of periodontitis, whereas the etiological role of the closely related but phenotypically indistinguishable species Prevotella nigrescens is controversial. To differentiate between these species properly, 16S rDNA/RNA directed, computer-optimized oligonucleotides were designed and tested with 26 P. intermedia, 26 P. nigrescens and a number of closely and more distantly related strains. The oligonucleotides were used as primers in a polymerase chain reaction and could be demonstrated to be species specific with a detection limit of 50 bacterial cells, which could also be detected when diluted 1:10(5) with different plaque bacteria. In addition, the described oligonucleotides were digoxigenin-labeled at the 3' end and used as DNA probes in a dot blot hybridization assay. This assay, although slightly less sensitive than the polymerase chain reaction-based method, gave species-specific reactions and also allowed (semi-)quantification of bacterial cells in clinical specimens.