Background: Hair is a promising tissue to assess exposure to 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), a carcinogen formed in cooked meats. However, an understanding of how dietary exposure to PhIP, cytochrome P450 1A2 activity-a key enzyme involved in PhIP metabolism, and hair pigmentation affect the level of PhIP accrued in hair is required to determine the reliability of the PhIP hair level as a biomarker of exposure to this carcinogen. Methods: We examined the impact of PhIP exposure, cytochrome P450 1A2 activity, and hair pigmentation on the levels of PhIP accumulated in the hair of volunteers on a 4-week semicontrolled diet of cooked meat containing known quantities of PhIP. Results: The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat. PhIP levels in hair were correlated to PhIP intake (r 0.53; P < 0.001), and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair (r 0.71; P < 0.001). However, PhIP accrual in hair was not correlated to cytochrome P450 1A2 activity, as assessed by the caffeine test, or to the levels of unmetabolized PhIP in urine or to the metabolic ratio of the major urinary metabolite N2-(b-1-glucosiduronyl-2- (hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine to unmetabolized PhIP. Conclusions: The use of the PhIP hair biomarker should take hair pigmentation into account for accurate exposure assessment of PhIP. Impact: PhIP hair levels can serve as a biomarker in epidemiologic studies investigating the association of heterocyclic aromatic amine (HAA), cooked meat, and cancer risk.