Although, 1,3-butadiene is a known human carcinogen emitted from mobile sources, little is known about traffic-related human exposure to this toxicant. This pilot study was designed to characterize traffic-related environmental exposure to 1,3-butadiene and evaluate its urinary mercapturic acids as biomarkers of exposure in these settings. Personal air samples and multiple urine samples were collected on two separate occasions from three groups of individuals that differed by spatial proximity as well as intensity of traffic: (i) toll collectors, (ii) urban-weekday and (iii) suburban-weekend group. Air samples were analyzed using thermal desorption followed by GC/MS and urine samples were analyzed using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) for two mercapturic acids of 1,3-butadiene: monohydroxy-3-butenyl mercapturic acid (MHBMA) and 1,2-dihydroxybutyl mercapturic acid (DHBMA). Exposure differed between groups (p<0.05) with median values of 2.38, 1.62 and 0.88 microg/m(3) for toll collectors, the urban-weekday group and the suburban-weekend group, respectively. A refined ID-LC-MS/MS method enabled detection of MHBMA, previously detected only in occupational settings, with high frequency. MHBMA and DHBMA were detected in 95 and 100% of urine samples at levels (mean+/-S.D.) of 9.7+/-9.5, 6.0+/-4.3 and 6.8+/-2.6 ng/mL for MHBMA and 378+/-196, 258+/-133 and 306+/-242 ng/mL for DHBMA for the three different groups, respectively. Mean biomarker levels were higher among the toll collectors compared to the other two groups, however, the differences were not statistically significant (p>0.05). This study is the first to evaluate 1,3-butadiene biomarkers for subtle differences in environmental exposures. However, additional research will be required to ascertain whether the lack of statistical association observed here is real or attributable to unexpectedly small differences in exposure between groups (<1 microg/m(3)), non-specificity of the biomarker at low exposure, and/or small sample size.