Cytochrome P450‐associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black‐crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3‐methylcholanthrene (200 μg administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle‐treated embryos), 3‐methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin‐O‐dealkylase, BROD; ethoxyresorufin‐O‐dealkylase, EROD; pentoxyresorufin‐O‐dealkylase, PROD) and a greater than 100‐fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000‐fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin‐O‐dealkylase (ECOD) were up to 85‐fold higher in pipping black‐crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross‐reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p′‐DDE were detected in embryos from Cat Island. Cytochrome P450‐associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50‐0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black‐crowned night heron embryos.