Anti-parkinsonian agents, pramipexole (PPX) and ropinirole (ROP), have been reported to possess neuroprotective properties, both in vitro and in vivo. The mechanisms underlying neuroprotection afforded by the D3-preferring receptor agonists remain poorly understood. The present study demonstrates that incubation of primary mesencephalic cultures with PPX and ROP or the conditioned medium from PPX- or ROP-treated primary cultures induced a marked increase in the number of dopamine (DA) neurons in the cultures. Similar effects can be observed after incubating with the conditioned medium derived from PPX- and ROP-treated substantia nigra astroglia. Meanwhile, PPX and ROP can protect the primary cells from insult of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Furthermore, the neurotrophic effects of PPX and ROP on mesencephalic dopamine neurons could be significantly blocked by D3 receptor antagonist, but not by D2 receptor antagonist. Moreover, we found that the levels of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the conditioned medium of mesencephalic cultures treated with PPX and ROP were significantly increased. Blocking GDNF and BDNF with the neutralizing antibodies, the neurotrophic effects of PPX and ROP were greatly diminished. These results suggest that D3 dopamine receptor-preferring agonists, PPX and ROP, exert neurotrophic effects on cultured DA neurons by modulating the production of endogenous GDNF and BDNF, which may participate in their neuroprotection.