Staphylococci represent the most commonly encountered blood culture isolates. Differentiating Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is important in guiding empirical therapy, especially since the majority of CoNS are contaminants. This study evaluated three rapid methods for the direct identification of S. aureus from blood cultures. A total of 157 patient blood cultures with gram stains showing gram-positive cocci in clusters were included. The following assays were evaluated: API RAPIDEC staph (API) (bioMerieux, Durham, N.C.), the tube coagulase test (TCT) read at 4 h, and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) (AdvanDx, Woburn, Mass.). All assays yielded results of S. aureus or non-S. aureus. The direct rapid results were compared to results obtained with isolated colonies using the AccuProbe Staphylococcus aureus Culture Identification Test (Gen-Probe, San Diego, Calif.). API, TCT, and PNA FISH exhibited sensitivities of 96, 84, and 99% and specificities of 99, 100, and 100%, respectively. Direct identification testing by any of these three assays yielded acceptable performance and timely results. This ability to accurately detect S. aureus in blood cultures gives the physician information with which to initiate or tailor antimicrobial therapy. Coupled with direct susceptibility testing of positive blood culture broths, the patient and institution may experience improved outcomes.