Experiments were conducted to assess the in vivo potency of binary mixtures of estrogenic chemicals using plasma vitellogenin (VTG) concentrations in juvenile rainbow trout (Oncorhynchus mykiss) as the endpoint. The estrogenic potencies of estradiol-17β (E2), 4-tert-nonylphenol (NP), and methoxychlor (MXC) were determined following 14 day exposures to the individual chemicals and binary mixtures of these chemicals. E2, NP, and MXC all induced concentration dependent increases in plasma VTG, with lowest observed effect concentrations of 4.7 and 7.9 ng L-1 for E2, 6.1 and 6.4 μg L-1 for NP, and 4.4 and 6.5 μg L-1 for MXC. Concentration−response curves for fixed ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1:1000), and NP and MXC (1:1) were compared to those obtained for the individual chemicals, using the model of concentration addition. Mixtures of E2 and NP were additive at the concentrations tested, but mixtures of E2 and MXC were less than additive. This suggests that while NP probably acts via the same mechanism as E2 in inducing VTG synthesis, MXC may be acting via a different mechanism(s), possibly as a result of its conversion to HPTE which is an estrogen receptor α agonist and an estrogen receptor β antagonist. It was not possible to determine whether mixtures of MXC and NP were additive using VTG induction, because the toxicity of MXC restricted the effect range for which the expected response curve for the binary mixture could be calculated. The data presented illustrate that the model of concentration addition can accurately predict effects on VTG induction, where we know that both chemicals act via the same mechanism in mediating a vitellogenic response.