Here, we report that leucine enkephalin (LE) is neuroprotective to dopaminergic (DA) neurons at femtomolar concentrations through anti-inflammatory properties. Mesencephalic neuron-glia cultures pretreated with femtomolar concentrations of LE (10(-15)-10(-13) M) protected DA neurons from lipopolysaccharide (LPS)-induced DA neurotoxicity, as determined by DA uptake assay and tyrosine hydroxylase (TH) immunocytochemistry (ICC). However, des-tyrosine leucine enkephalin (DTLE), an LE analogue that is missing the tyrosine residue required for binding to the kappa opioid receptor, was also neuroprotective (10(-15)-10(-13) M), as determined by DA uptake assay and TH ICC. Both LE and DTLE (10(-15)-10(-13) M) reduced LPS-induced superoxide production from microglia-enriched cultures. Further, both LE and DTLE (10(-14), 10(-13) M) reduced the LPS-induced tumor necrosis factor-alpha (TNFalpha) mRNA and TNFalpha protein from PHOX+/+ microglia, as determined by quantitative real-time RT-PCR and ELISA analysis in mesencephalic neuron-glia cultures, respectively. However, both peptides failed to inhibit TNFalpha expression in PHOX-/- cultures, which are unable to produce extracellular superoxide in response to LPS. Additionally, LE and DTLE (10(-14), 10(-13) M) failed to show any neuroprotection against LPS in PHOX-/- cultures. Together, these data indicate that LE and DTLE are neuroprotective at femtomolar concentrations through the inhibition of oxidative insult associated with microglial NADPH oxidase and the attenuation of the ROS-mediated amplification of TNFalpha gene expression in microglia.