Background: In 2002, the oil tanker Prestige spilled more than 67 000 tons of bunker oil, heavily contaminating the coast of northwestern Spain. Objective: To assess respiratory effects and chromosomal damage in clean-up workers of the oil spill 2 years after the exposure. Design: Cross-sectional study. Setting: Fishermen cooperatives in coastal villages. Participants: Local fishermen who were highly exposed (n = 501) or not exposed (n = 177) to oil 2 years after the spill. Measurements: Respiratory symptoms; forced spirometry; methacholine challenge; markers of oxidative stress (8-isoprostane), airway inflammation (interleukins, tumor necrosis factor-α, and interferon-γ), and growth factor activity in exhaled breath condensate; and chromosomal lesions and structural alterations in circulating lymphocytes. Results: Compared with nonexposed participants, persons exposed to oil were at increased risk for lower respiratory tract symptoms (risk difference, 8.0 [95% CI, 1.1 to 14.8]). Lung function did not significantly differ between the groups. Among nonsmoking participants, exposed individuals had higher exhaled 8-isoprostane levels than nonexposed individuals (geometric mean ratio, 2.5 [CI, 1.7 to 3.7]), and exposed individuals with lower respiratory tract symptoms had higher 8-isoprostane levels than those of exposed individuals without symptoms. Exposed nonsmoking participants also had higher levels of exhaled vascular endothelial growth factor (risk difference, 44.8 [CI, 27.9 to 61.6]) and basic fibroblast growth factor (risk difference, 16.0 [CI, 3.5 to 28.6]). A higher proportion of exposed participants had structural chromosomal alterations (risk difference, 27.4 [CI, 10.0 to 44.8]), predominantly unbalanced alterations. The risk for elevated levels of exhaled 8-isoprostane, vascular endothelial growth factor, and basic fibroblast growth factor and structural chromosomal alterations seemed to increase with intensity of exposure to clean-up work. Limitations: The clinical significance of exhaled biomarkers and chromosomal findings are uncertain. The association between oil exposure and the observed changes may not be causal. The findings may not apply to spills involving other types of oil or to different populations of oil spill workers. Conclusion: Participation in clean-up of a major oil spill was associated with persistent respiratory symptoms, elevated markers of airway injury in breath condensate, and chromosomal damage. Primary Funding Source: Instituto de Salud Carlos III. © 2010 American College of Physicians.