J. Neurochem. (2010) 114, 843–852. Abstract The aim of the present study was to investigate whether late pre‐conditioning using 3‐nitropropionic acid (3NP) prevents the 5‐hydroxytryptamine (5‐HT) deficits caused by the amphetamine derivative 3,4‐methylenedioxymethamphetamine (MDMA) in the rat. For this purpose we administered 3NP 24 h before MDMA (3 × 5 mg/kg i.p., every 2 h) and rats were killed 7 days later. Pre‐treatment of 3NP afforded complete protection against MDMA‐induced 5‐HT deficits independent of any effect on MDMA‐induced hyperthermia or 5‐HT transporter activity. To identify the transductional mechanisms responsible for the neuroprotective effect of 3NP, we first examined the involvement of nitric oxide (NO) by using selective inhibitors of all three nitric oxide synthase isoforms. Inhibition of endothelial and neuronal nitric oxide synthase, but not inducible nitric oxide synthase, reversed 3NP‐induced pre‐conditioning. The NO donor S‐Nitroso‐N‐acetylpenicilamine mimicked 3NP effects further suggesting the involvement of NO in mediating 3NP protection. To investigate the involvement of NOS/soluble guanylate cyclase (sGC)/protein kinase G/mitochondrial ATP‐sensitive potassium channels (mitoKATP) signaling pathway we examined the effect of 5‐hydroxydecanoate (5‐HD), a selective mitoKATP blocker, and 1H‐(1,2,4)oxadiazolo[4,3‐a]quinoxaline‐1‐one, a potent inhibitor of sGC, on 3NP‐induced tolerance. 5‐hydroxydecanoate, but not 1H‐(1,2,4)oxadiazolo[4,3‐a]quinoxaline‐1‐one, suppressed 3NP‐mediated protection suggesting that mitoKATP opening, but not NO‐mediated activation of sGC, participates in the mechanism underlying tolerance to MDMA. Our data also showed that the protective effect of 3NP was abolished by cycloheximide, supporting the involvement of de novo protein synthesis. In conclusion, 3NP‐induced delayed tolerance against 5‐HT deficits caused by MDMA occurs via NO production.