Thyroid hormone (TH) is essential for normal brain development, and polychlorinated biphenyls (PCBs) are known to interfere with TH action in the developing brain. Thus, it is possible that the observed neurotoxic effects of PCB exposure in experimental animals and in humans are mediated in part by their ability to interfere with TH signaling. PCBs may interfere with TH signaling by reducing circulating levels of TH, by acting as TH receptor (TR) analogues, or both. If PCBs act primarily by reducing serum TH levels, then their effects should mimic those of low TH. In contrast, if PCBs act primarily as TH agonists in the developing brain, then they should mimic the effect of T₄ in hypothyroid animals. We employed a two-factor design to test these predictions. Both hypothyroidism (Htx) and/or PCB treatment reduced serum free and total T₄ on postnatal day 15. However, only Htx increased pituitary TSH expression. RC3/Neurogranin expression was decreased by Htx and increased by PCB treatment. In contrast, PCP-2 expression was reduced in hypothyroid animals and restored by PCB treatment. Finally, PCB treatment partially ameliorated the effect of Htx on the thickness of the External Granule layer (EGL) of the cerebellum. These studies demonstrate clearly that PCB exposure does not mimic the effect of low TH on several important TH-sensitive measures in the developing brain. However, neither did PCBs mimic T₄ in hypothyroid animals on all endpoints measured. Thus, PCBs exert a complex action on TH signaling in the developing brain.