Presently, few biomarker-based approaches are available for the evaluation of environmental exposure to persistent organic pollutants in dairy ruminants. In this study, goats (Capra hircus) were orally administered a mixture of pyrene, phenanthrene, and benzo[a]pyrene daily over a 40-d period (1 or 50 mg/d). Milk and urine 1-hydroxypyrene levels, ethoxyresorufin-O-deethylase (EROD) activity in peripheral blood lymphocytes (PBL) as well as urinary levels of 2- and 3-hydroxyphenanthrene were determined at 10-d intervals. 1-Hydroxypyrene excretion in milk and urine significantly increased and then achieved a plateau at 10 d. Transfer rates of 1-hydroxypyrene were calculated to be approximately 0.5 and 25% in milk and urine, respectively. Concentrations in milk and urine were proportional to the ingested doses. These results demonstrate that 1-hydroxypyrene in milk or urine may be used as a biomarker for evaluating the exposure of dairy ruminants to polycyclic aromatic hydrocarbons (PAHs) over an extended exposure period. Constitutive EROD activity in lymphocytes was 0.5 +/- 0.3 pmol resorufin/min/mg protein, and was significantly induced over the entire exposure time, before stabilizing after 40d at 6.30 +/- 1.3 and 18.89 +/- 1.12 pmol resorufin/min/mg protein for 1 mg/d and 50 mg/d doses, respectively. Induction kinetics were calculated using a logistic-like model and approximate dose-response curves were designed. We therefore propose EROD activity in PBL as a relevant, convenient, and noninvasive biomarker of subchronic exposure of dairy ruminants to CYP450 inducing PAH. Environ. Toxicol. Chem. 2011;30:1346-1353. (C) 2011 SETAC