Cell replacement therapy using mesencephalic precursor cells is an experimental approach for the treatment of Parkinson's disease (PD). A significant problem associated with this procedure is the poor survival of grafted neurons. Impaired energy metabolism is considered to contribute to neuronal cell death after transplantation. Creatine is a substrate for mitochondrial and cytosolic creatine kinases (CK) and buffers cellular ATP resources. Furthermore, elevated cellular creatine levels facilitate metabolic channeling and show antiapoptotic properties. Exogenous creatine supplementation therefore might offer a tool for improvement of dopaminergic neuron survival. The present study aimed at investigating the effects of creatine on cell survival of rat embryonic day 14 (E14) ventral mesencephalic neurons grown as organotypic free-floating roller tube (FFRT) cultures. We found that the brain-specific isoform of CK (BB-CK) and the ubiquitous mitochondrial isoform (uMt-CK) are expressed at high levels in FFRT cultures and colocalize with tyrosine hydroxylase immunoreactive (TH-ir) cells. Exposure of these cultures to creatine induced an increase in the content of the BB-CK isotype. Creatine (5 mM) administration starting at day in vitro (DIV) 7 resulted in a significant increase (+35%) in TH-ir cell density at DIV21. In addition, we observed that creatine treatment provided neuroprotection against 1-methyl-4-phenyl pyridinium ion (MPP+)-induced TH-ir cell loss in the FFRT culture system, resulting in a significantly higher density (+19%) of TH-ir neurons in creatine-treated cultures compared to corresponding controls. The decrease of TH-ir neurons in the MPP+-treated group corresponded with an increase in immunoreactivity for active caspase-3, an effect that was not seen in the group receiving creatine supplementation. In conclusion, our data imply that creatine administration is beneficial for the survival of TH-ir neurons encountering harmful conditions.