We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1-methyl-4-phenylpyridinium (MPP+) and glial derived neurotrophic factor (GDNF) to validate this novel culture model. Dopamine production and dopaminergic neuron soma size increased dramatically with time in vitro, whereas the number of dopamine neurons declined by approximately 30% between week 1 and week 2, which was further reduced after week 4. GDNF treatment (100 ng/mL) increased dopaminergic neuron soma size (up to 43%) and DOPAC production (approximately three-fold), but not the number of dopamine neurons in control cultures. One-week-old cultures were more vulnerable to MPP+, than three-week-old cultures. The EC50 for dopamine depletion after 2 days exposure and 15 days of recovery were 0.6 and 7 µm, respectively. Both pre-treatment and post-treatment with GDNF are important to obtain maximal protection against MPP+ toxicity. In one-week-old cultures (5 µm MPP+, 2 days) GDNF provided potent neuroprotection with dopamine contents reaching control levels and number of tyrosine hydroxylase (TH)+ cells up to 80% of control, but in three-week-old cultures (10 µm MPP+, 2 days) the protective potential of GDNF was markedly reduced. Long recovery periods after MPP+ exposure are required to distinguish between reversible or irreversible toxic and/or trophic effects.