Recent immunohistochemical studies of rat brain triiodothyronine reveal heaviest localization in locus coeruleus perikarya. The cellular distribution is similar to that observed in concomitant studies of tyrosine hydroxylase immunohistochemistry: heavy clumps of immunoreactive triiodothyronine are distributed within locus coeruleus cytosol and in cell processes, leaving cell nuclei unstained. At the same time, in locus coeruleus targets, cell nuclei as well as surrounding neuropil are prominently triiodothyronine labeled. These observations, combined with diverse evidence linking thyroid hormone with norepinephrine at many levels of physiological and pathophysiological function, led to the hypothesis that the locus coeruleus binds and accumulates triiodothyronine and delivers the hormone via anterograde axonal transport to postsynaptic locus coeruleus targets, where nuclear triiodothyronine receptors are densely concentrated. Furthermore, the hypothesis predicts that destruction of locus coeruleus nerve terminals would interrupt this neural route of triiodothyronine delivery and prevent or reduce triiodothyronine labeling of nuclear receptors in noradrenergic target cells. To test this formulation, we gave the specific locus coeruleus lesioning agent, N-(2-chloroethyl)-N-2-bromobenzylamine hydrochloride (DSP-4), to adult male rats and examined their brains three, five and seven days thereafter by triiodothyronine and, in alternate sections, tyrosine hydroxylase immunohistochemistry. Treatment with DSP-4 resulted in specific and selective reduction in tyrosine hydroxylase and triiodothyronine immunohistochemical labeling in cell nuclei and in nerve cell processes within the neuropil of the hippocampus and cerebral cortex at all time periods examined. The results demonstrate that full occupancy of locus coeruleus target cells by triiodothyronine requires the presence of intact locus coeruleus projections and supports the proposal that, like norepinephrine, triiodothyronine delivery to noradrenergic targets occurs through delivery by locus coeruleus terminals. These findings provide strong support for earlier proposals that triiodothyronine functions as a co-transmitter with norepinephrine in addition to or as part of its genomic role in the cells receiving noradrenergic innervation.