The elevation of the two genotypes of Prevotella intermedia to species rank as P. intermedia and Prevotella nigrescens has increased the need for reliable differentiation between the two taxa. In this study, 53 strains, including strains whose species affiliations were known as well as fresh dental plaque isolates, were subjected to a multilocus enzyme electrophoretic analysis, DNA analyses in which we used whole genomic DNA, rRNA sequences, and an oligonucleotide specific for the former P. intermedia genotype II (P. nigrescens) as probes, and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of soluble cellular proteins. All of these tests consistently separated the strains into the same two distinct groups corresponding to P. intermedia and P. nigrescens, confirming that the two species constitute two distinct populations of bacteria. Each of the tests used independently provided reliable identification to the species level. A previously reported heterogeneity in the pattern of human immunoglobulin A1 (IgA1) degradation was not confirmed. No differences between species were observed. All of the strains induced total degradation of IgA1 within 48 h, a property that may be a virulence factor in periodontal disease development. The enzymes responsible for IgA1 degradation were not inactivated by the physiological proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor.