Objective: To evaluate the relative contribution of occupational vs. non-occupational secondhand tobacco smoke exposure to overall hair nicotine concentrations in non-smoking bar and restaurant employees. Method: We recruited 76 non-smoking employees from venues that allowed smoking (n=9), had mixed policies (smoking and non-smoking areas, n=13) or were smoke-free (n=2) between April and August 2008 in Santiago, Chile. Employees used personal air nicotine samplers during working and non-working hours for a 24-h period to assess occupational vs. non-occupational secondhand tobacco smoke exposure and hair nicotine concentrations to assess overall secondhand tobacco smoke exposure. Results: Median hair nicotine concentrations were 1.5ng/mg, interquartile range (IQR) 0.7 to 5.2ng/mg. Time weighted average personal air nicotine concentrations were higher during working hours (median 9.7, IQR 3.3-25.4μg/m3) compared to non-working hours (1.7, 1.0-3.1μg/m3). Hair nicotine concentration was best predicted by personal air nicotine concentration at working hours. After adjustment, a 2-fold increase in personal air nicotine concentration in working hours was associated with a 42% increase in hair nicotine concentration (95% confidence interval 14-70%). Hair nicotine concentration was not associated with personal air nicotine concentration during non-working hours (non-occupational exposure). Conclusions: Personal air nicotine concentration at working hours was the major determinant of hair nicotine concentrations in non-smoking employees from Santiago, Chile. Secondhand tobacco smoke exposure during working hours is a health hazard for hospitality employees working in venues where smoking is allowed.