Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Gram's stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.