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The class I fructose 1,6-diphosphate aldolases of five staphylococcal strains were purified and characterized. All of these enzymes could be renatured to active structures after exposure to a temperature of 90°C. By using antisera against the enzymes, we measured the immunological cross-reactions by microcomplement fixation and activity inhibition. The amino acid compositions of the purified proteins were determined and compared. Our results provide further insight into the phylogenetical relationships among the staphylococcal species studied. Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus warneri, and Staphylococcus haemolyticus form a group of highly related species. Staphylococcus aureus was shown to be closely related to this group, whereas all of the other staphylococcal species showed distant relationships. Staphylococcus sciuri subsp. sciuri and S . sciuri subsp. lentus occupy a rather isolated position within the genus Staphylococcus and are related to each other at the species level.The fructose 1,6-diphosphate (FDP) aldolases which are usually found in bacteria and fungi are class I1 enzymes (31). These enzymes need a divalent metal ion for activity and are inhibited by chelating agents. In contrast, class I aldolases have been found in most staphylococci (9) and a few other bacteria (13, 20, 21, 35). Class I aldolases can be irreversibly inhibited by dihydroxyacetone phosphate in the presence of sodium borohydride (12). The class I FDP aldolases from staphylococci and peptococci appear to be similar in enzymatic kinetics and molecular weight. The presence of class I aldolases in staphylococci is a useful property for distinguishing these organisms from other members of the family Micrococcaceae (9). Both class I1 aldolase activity and class I aldolase activity have been found in Staphylococcus hyicus and Staphylococcus intermedius strains, but only a class I1 enzyme occurs in Staphylococcus caseolyticus (6). This difference provides a simple method for classifying these staphylococci within the genus. Furthermore, the phylogenetic relationships of staphylococci have been determined by deoxyribonucleic acid (DNA)-DNA (15, 17, 24, 29, 32) and DNA-ribonucleic acid (RNA) homology (14) studies, as well as by comparing 16s ribosomal RNAs (23) and immunological cross-reactions of catalases (30,32). In the present study we used the following two essentially different methods to reveal the phylot Present address: