Three hundred and twenty isolates of Staphylococcus aureus were typed by DNA sequence analysis of the X region of the protein A gene (spa). spa typing was compared to both phenotypic and molecular techniques for the ability to differentiate and categorize S. aureus strains into groups that correlate with epidemiological information. Two previously characterized study populations were examined. A collection of 59 isolates (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hébert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407–415, 1994) from the Centers for Disease Control and Prevention (CDC) was used to test for the ability to discriminate outbreak from epidemiologically unrelated strains. A separate collection of 261 isolates form a multicenter study (R. B. Roberts, A. de Lencastre, W. Eisner, E. P. Severina, B. Shopsin, B. N. Kreiswirth, and A. Tomasz, J. Infect. Dis. 178:164–171, 1998) of methicillin-resistant S. aureus in New York City (NYC) was used to compare the ability of spa typing to group strains along clonal lines to that of the combination of pulsed-field gel electrophoresis and Southern hybridization. In the 320 isolates studied, spa typing identified 24 distinct repeat types and 33 different strain types. spa typing distinguished 27 of 29 related strains and did not provide a unique fingerprint for 4 unrelated strains from the four outbreaks of the CDC collection. In the NYC collection, spa typing provided a clonal assignment for 185 of 195 strains within the five major groups previously described. spa sequencing appears to be a highly effective rapid typing tool for S. aureus that, despite some expense of specificity, has significant advantages in terms of speed, ease of use, ease of interpretation, and standardization among laboratories.