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Urinary 1,6-hexamethylene diamine (HDA) has been indicated as a potential biomarker for systemic exposure to 1,6-hexamethylene diisocyanate (HDI). Our objective was to establish a quantitative and time-dependent relationship between urine HDA levels and inhalation and dermal exposure to HDI. Urine HDA levels, creatinine concentrations, and specific gravity along with dermal and breathing-zone levels of HDI were measured in automotive spray painters. Dermal exposure and inhalation exposure levels, adjusted for the type of respirator worn, and creatinine concentration were observed to be significant predictors of urine HDA levels. The use of coveralls, type of respirator worn, smoker status, type of painting booth, and multiple painters at the shop were found to significantly affect urine HDA. Our results indicate that urine HDA is a useful biomarker for systemic HDI exposure through the skin and lungs and emphasize the need for proper dermal and inhalation protection for workers.