No typing system exists for Aspergillus fumigatus, though isolates are distinguishable by phenotypic characteristics. DNA was prepared by lysis of protoplasts, followed by deproteination, phenolchloroform extraction, and dialysis. DNA prepared was of uniform size and exceeded 60 kb. After digestion with SalI and XhoI endonucleases, DNA was electrophoresed, stained, and photographed. Differences in the mobilities of 10- to 50-kb bands distinguished isolates. Reproducibility was shown by repeated preparations and animal passage. By use of a proposed notation system for describing restriction fragment length polymorphism patterns, 31 epidemiologically characterized isolates from three continents revealed 24 patterns (DNA types). Three DNA types were represented by 3 isolates each and 1 DNA type by 2 isolates; 20 types were unique. Two groups of 3 isolates of the same DNA type were from Stanford University Hospital. One patient isolate from Stanford was the same DNA type as a sewage isolate from New Jersey. Another Stanford isolate was the same as a German isolate. These observations indicate widespread dispersal of some clones and restricted locales for others. Paired isolates from airway fluids of three patients had two DNA types in each. Restriction endonuclease typing shows promise for investigating the epidemiology and ecology of A. fumigatus.