Lysis of peptidoglycans of Staphylococcus aureus yields products that exhibit strain-specific patterns on analysis by thin-layer chromatography and polyacrylamide gel electrophoresis. The patterns are demonstrated when products of the endogenous autolysins are examined and are enhanced and/or altered by the use of exogenous murolytic enzymes. These fingerprinting techniques were applied to the study of 55 strains of S. aureus. Twenty-six strains were from patients in five localized hospital outbreaks, and 29 were obtained randomly from individual patients. With only two exceptions all strains from a given outbreak had an identical murolytic pattern, whereas the random strains showed variable patterns. Peptidoglycan fingerprinting may be an adjunct tool for the hospital epidemiologist and may also be valuable for the study of certain biologic aspects of interstrain differences.