Staphylococcus aureus produces multiple bacteriolytic enzymes (autolysins) and grows usually as a mixture of single cells, pairs, short chains, and irregular clusters, Autolysin-defective mutants that form cubic cell packets (Pa4A and PaH13) or grape-like clusters (Cu9S and CuD10) were isolated from S. aureus FDA 209P after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The Pa4A mutant grown in nutrient broth formed cell packets consisting of 8-64 cells that appeared regularly arranged in three dimensions. Thin-section electron micrographs revealed that the packet cells were encased in an orderly manner within a thick peripheral wall and that their septa failed to split, Zymographic analysis of enzyme extracts from mutant Pa4A showed that it lacked the 33-kDa autolytic enzyme band present in the parent strain. Another mutant, Cu9S, formed grape-like clusters and showed a single autolytic enzyme band (33-kDa). The possibility that the 33-kDa autolytic enzyme is involved in splitting of the septum prior to cell separation in S. aureus is discussed.