Risk factors have not been identified that determine susceptibility for development of diisocyanate-induced occupational asthma (DA). We hypothesized that diisocyanate (DI) exposure could modify gene promoter regions regulating transcription of cytokine mediators and thereby influence expression of DA. A cross-sectional study was designed to investigate the promoter methylation status of candidate genes in DI-exposed workers. Subjects consisted of 131 workers in three groups: 40 cases with DA confirmed by a positive specific inhalation challenge (SIC) (DA+), 41 exposed workers with lower respiratory symptoms and negative SIC (DA-), and 50 asymptomatic exposed workers (AWs). We studied four candidate genes (GSTM1, DUSP22, IFN-γ, and IL-4) for which altered promoter methylation has been previously investigated for relationships with a variety of other environmental exposures. Methylation status was determined using methylation-specific quantitative PCR performed on genomic DNA extracted from whole blood. Results showed that relative methylation of IFN-γ promoter was significantly increased in DA+ in comparison with both comparator groups (DA- and AW), and it exhibited good sensitivity (77.5%) and specificity (80%) for identifying DA workers in a multivariate predictive model after adjusting for type of DI exposure, smoking status, methacholine PC₂₀, and gender. IL-4 promoter was slightly less methylated only in DA+ compared with AW among nonsmoking workers. Both GSTM1 and DUSP22 promoter methylations were found not associated with DA. Our finding suggests that exposure to occupational chemicals could play a heretofore undefined mechanistic role via epigenetic modification of specific genes in the promoter region.