In order to biomonitor lead contamination in Southeastern Spain, 218 blood samples from 28 to 30-day old Eurasian Eagle Owl chicks (Bubo bubo) born between 2003 and 2007 were analysed. In general, mean lead levels showed that chicks were exposed to background concentrations. However, mean levels in chicks born in an ancient and abandoned mining site ("Sierra Minera Cartagena-La Union") or in their surroundings (Geometric mean (GM) = 5.83 μg/dl, range 0.49-25.61 μg/dl), an area highly polluted by lead and other metals, were significantly higher (p < 0.001) than the rest of the population (GM = 1.66 μg/dl, range = Non detected-18.37 μg/dl). Because δ-ALAD activity is considered the best biomarker for lead exposure and effect in birds, the activity of this enzyme was also evaluated and correlated with lead levels in blood. In this study, low levels of blood lead inhibited δ-ALAD, even when lead concentrations were lower than the limits described by other authors in raptors. Adverse effects caused by this inhibition may occur when blood lead levels were above 15 μg/dl, although only eight chicks presented these concentrations in their blood. Sampling site also influenced enzymatic activity, since it decreased about 60% in the polluted area in relation to the rest. For all these reasons, further research regarding risk assessment for lead exposure in Eagle Owls nesting in the polluted area is advisable. Our results suggest that the Eurasian Eagle Owl can be considered a suitable sentinel animal for monitoring lead contamination and δ-ALAD activity can be used as a sensitive biomarker for lead exposure and effect in this species.