Our group and others have shown that a single, universal primer pair designed to anneal to highly conserved regions within the bacterial 16S ribosomal RNA coding sequence (16S rDNA) can amplify genomic DNA from a wide variety of bacterial organisms. Since the highly conserved regions of the 16S rDNA flank regions of the gene which vary between species, we proposed to determine whether products amplified with a universal primer pair could then be used for identification of the species with specific hybridization probes. We examined a variety of bacterial 16S rDNA sequences from GenEMBL, a non-overlapping nucleotide sequence database, using FastA (GCG, Madison, WI). These sequences were aligned using PileUp (GCG, Madison, WI) and 19-21 bp sequences were identified with apparent species specificity. PCR amplification was performed with the universal primer pair, p806R and p8FPL, and amplification products were evaluated by manual and automated methods. Dot blots were prepared with the PCR products from both Gram positive and Gram negative organisms and hybridized with end-labeled species specific probes. Using this approach we were able to differentiate between PCR products generated from different bacterial DNA templates. We conclude, that individual species can be identified by species specific sequence variability within amplification products generated following PCR with a universal bacterial primer pair. We speculate that the application of molecular genetic technology for the diagnosis of bacterial infections will reduce the cost and the time for identification of pathogens, will allow for the molecular triage of sepsis, and will improve the quality of care for patients with infections.(E.R.B. McCabe is an Academic Associate for Corning-Nichols Institute.)