Methotrexate is a therapeutic agent used widely for osteosarcoma. We used an extremely sensitive high-performance liquid-chromatography assay to evaluate 112 urine samples obtained from 28 hospital employees during high-dose therapy with methotrexate and during routine care of patients. The highest cumulative urinary excretion was observed when methotrexate infusions were handled in a workbench from which a portion of filtered air was emitted into the room. Remarkable urine contaminations were identified for personnel, including 1 administrative employee who had "stood by" for 2 h in the room where infusions were prepared. Lower methotrexate concentrations were detected in the urine of nurses whose exclusive function was to care for patients. The urine burden in oncologic nurses decreased after a central pharmacy unit was installed. Methotrexate was excreted in the sweat of patients who were under high-dose therapy, and its elimination half-life was 11.1 h (mean maximal concentration = 1.7 micrograms/ml [n = 51). The maximal burden in spontaneous vomit from these patients was 441.5 micrograms/ml, and it declined to 0.24 micrograms/ml 19.5 h after infusion was completed. No methotrexate was detected in personnel who prepared 20-g methotrexate infusions in the central pharmacy unit. We demonstrated that occupational safety depended not only on technical precautions, but on the skills of specifically trained personnel.