Urinary 1-hydroxypyrene (1-OH-pyrene) is now largely considered to be a valuable biomarker of exposure of man and animals to pyrene and other polycyclic aromatic hydrocarbons (PAHs). However, from a practical and agronomic standpoint, the question remains whether such biomarking capability still holds when 1-OH-pyrene is analyzed in milk produced by ruminants. To assess this hypothesis, four goats were daily submitted to three different amounts of pyrene oral ingestion, together with phenanthrene and benzo(a)pyrene (1, 7, and 49 mg/day during 1 week each). An HPLC-fluorometric analysis of 1-OH-pyrene in milk revealed a perfect correlation between pyrene doses and 1-OH-pyrene detected in milk, thus fully confirming the biomarking capability of 1-OH-pyrene and providing information on its transfer coefficient toward milk. Transfer equations such as the ones found in the present study could be used as a valuable and practical risk assessment tool in (i) the accurate monitoring of exposure of ruminants to pyrene and (ii) the evaluation of occupational and environmental exposure of ruminants to PAH mixtures.