The role of ATP as a fast neurotransmitter is emerging from several lines of physiological and pharmacological studies. The bulk of experimental data on release properties and purinergic receptor-mediated postsynaptic potentials derives from studies in the habenula, but the source of the stimulation-evoked ATP release in this region is still unknown. In the present study, retrograde and anterograde tracing techniques were used to establish that both calretinin-containing and calretinin-negative neurons in the triangular septal and septofimbrial nuclei send a massive projection to the medial habenula, where they form asymmetrical synapses with their target neurons. The cells of origin, their axon terminals, as well as their synaptic targets remained unstained in sections immunostained for GABA. Electrolytic lesions of this anatomically circumscribed pathway resulted in an over 80% decrease in ATP release from habenula slices evoked by electric field stimulation. The possibility of transneuronal effects and release from local collaterals of habenular projection neurons accounting for the decreased ATP release has been excluded, since (i) there were no signs of neuronal degeneration, chromatolysis or atrophy in the habenula, (ii) the projection neurons have extremely sparse local collaterals and (iii) there are apparently no interneurons in the habenula. We conclude that the projection from the triangular septal and septofimbrial nucleus to the habenula uses ATP as a fast neurotransmitter, and its co-transmitter, if any, is likely to be glutamate.