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After five patients were diagnosed with nosocomial invasive aspergillosis caused by Aspergillus fumigatus and A. flavus, a 14-month surveillance program for pathogenic and nonpathogenic fungal conidia in the air within and outside the University Hospital in Rotterdam (The Netherlands) was begun. A. fumigatus isolates obtained from the Department of Hematology were studied for genetic relatedness by randomly amplified polymorphic DNA (RAPD) analysis. This was repeated with A. fumigatus isolates contaminating culture media in the microbiology laboratory. The density of the conidia of nonpathogenic fungi in the outside air showed a seasonal variation: higher densities were measured during the summer, while lower densities were determined during the fall and winter. Hardly any variation was found in the numbers of Aspergillus conidia. We found decreasing numbers of conidia when comparing air from outside the hospital to that inside the hospital and when comparing open areas within the hospital to the closed department of hematology. The increase in the number of patients with invasive aspergillosis could not be explained by an increase in the number of Aspergillus conidia in the outside air. The short-term presence of A. flavus can only be explained by the presence of a point source, which was probably patient related. Genotyping A. fumigatus isolates from the department of hematology showed that clonally related isolates were persistently present for more than 1 year. Clinical isolates of A. fumigatus obtained during the outbreak period were different from these persistent clones. A. fumigatus isolates contaminating culture media were all genotypically identical, indicating a causative point source. Knowledge of the epidemiology of Aspergillus species is necessary for the development of strategies to prevent invasive aspergillosis. RAPD fingerprinting of Aspergillus isolates can help to determine the cause of an outbreak of invasive aspergillosis.