Accumulation of four 14C‐labeled polychlorinated dibenzo‐p‐dioxins (PCDDs), 1,2,3,7‐T4CDD, 1,2,3,4,7‐P5CDD, 1,2,3,4,7,8‐H6CDD, and 1,2,3,4,6,7,8‐H7CDD, by juvenile rainbow trout (Salmo gairdneri) and fathead minnows (Pimephales promelas) was studied by feeding the fish treated food for 30 d followed by a 30‐ to 75‐d depuration period. Assimilation efficiencies (based on total 14C) of the isomers were low, ranging from 13% for H7CDD to 37% for H6CDD in trout, as compared with 64% for 4,4′‐DDT under the same conditions. Bioaccumulation factors, assimilation efficiencies and elimination rate constants for each PCDD congener were lower in fathead minnows than in rainbow trout. Half‐lives for total 14C ranged from 9 to 13 d for T4CDD and P5CDD in trout and minnows, respectively, and from 46 to 112 d for H6CDD and H7CDD in the same fish. Toluene‐extractable radioactivity for T4CDD and P5CDD was less than 30% during the clearance phase, indicating extensive transformation to polar products in both species. A four‐compartment model was used to estimate the accumulation of H6CDD and H7CDD in Great Lakes food chains (from detritus/phytoplankton to lake trout). The model predicted decreasing concentrations of the PCDDs at higher trophic levels, with accumulation primarily from food, assuming freely dissolved water concentrations of less than 2 × 10−5 ng L−1.