L-triiodothyronine (T3) has previously been shown to enhance fast-phase, depolarization-induced 45Ca uptake and 3H-gamma-aminobutyric acid release by rat brain synaptosomes at low nanomolar concentrations comparable to those reported for whole brain. Nevertheless, the physiologic importance of these nonnuclear-mediated effects of T3 has remained uncertain, in part because specific mechanisms and the presence of T3 at presumptive sites of action have not been demonstrated. Isotopic studies showing that L-tetraiodothyronine (thyroxine T4) and T3 are concentrated in synaptosomes, and that T4 is deiodinated to T3 suggested that endogenous levels of T3 in nerve terminals are probably much higher than in other compartments of the brain. In the present study we confirmed that endogenous levels of T3 in nerve terminals are at least eightfold higher, and may be as much as 60-fold higher, than in whole brain. More importantly, we showed that both 125I-labeled T3 and endogenous T3, but not 125I-T4 or endogenous T4, are released from depolarized synaptosomes, primarily by a Ca(2+)-dependent process. This demonstrates a mechanism for raising the level of T3 within the synapse, where the hormone may interact with pre- and postsynaptic binding (or uptake) sites, and suggests that the peripheral hormone T3 may be a neurotransmitter.