In previous studies, 3 linkage groups of genetic loci were constructed by transformation analyses in S. aureus NCTC 8325. In this study, interpretations of protoplast fusion data accumulated from 9- and 10-factor crosses have allowed the arrangement of the 3 linkage groups as a circular map. Fusion analysis also was successful in predicting the locations of unmapped chromosomal markers. The computerized analysis of 9- and 10-factor crosses allowed the user to score and enter the data directly into the computer from replica plates. The data were then condensed into an array of phenotypes along with their observed frequencies. Further analysis included the calculation of marker-marker coinheritance frequencies. The interpretations of the fusion data were entirely consistent with transformation-derived marker relationships. Linkage of (OMEGA)34Chr::Tn551 of linkage group III and tyrB282::Tn551 ermB321 of linkage group I, and the positions of 3 orphan markers were both predicted by the results of protoplast fusions. A procedure for extracting high molecular weight transforming DNA from protoplasts was developed and used to confirm the predicted linkages. Protoplast fusion analysis followed by quantitative transformation analysis with high molecular weight DNA was found to be a rapid and efficient way of mapping chromosomal markers in S. aureus.