Our objectives were to determine relative contributions of diet and dust exposure from multiple microenvironments to PentaBDE body burden, and to explore the role of handwipes as a measure of personal exposure to PentaBDE. We administered a food frequency questionnaire and collected serum, dust (office, main living area, bedroom, and vehicle), and handwipe samples from 31 participants. ΣPentaBDEs (sum of BDE 28/33, 47, 99, 100, and 153) in handwipes collected in the office environment were weakly correlated with dust collected from offices (r = 0.35, p = 0.06) and bedrooms (r = 0.39, p = 0.04), but not with dust from main living areas (r = -0.05, p = 0.77) or vehicles (r = 0.17, p = 0.47). ΣPentaBDEs in serum were correlated with dust from main living areas (r = 0.42, p = 0.02) and bedrooms (r = 0.49, p = 0.008), but not with dust from offices (r = 0.22, p = 0.25) or vehicles (r = 0.20, p = 0.41). Our final regression model included variables for main living area dust and handwipes, and predicted 55% of the variation in serum ΣPentaBDE concentrations (p = 0.0004). Diet variables were not significant predictors of ΣPentaBDEs in serum. Our research suggests that exposure to dust in the home environment may be the most important factor in predicting PentaBDE body burden in North Americans, and potential exposure pathways may involve PBDE residues on hands.