Progesterone synthesis by the human placenta requires the conversion of mitochondrial cholesterol to pregnenolone by cytochrome P450scc. Most steroidogenic tissues use the steroidogenic acute regulatory protein ( StAR) to deliver cholesterol to the inner mitochondrial membrane where P450scc is located, but StAR is not expressed in the human placenta. However, the human placenta does express MLN64, which has a C-terminal domain homologous to StAR that can also transport cholesterol. We investigated the ability of bacterially expressed N-218 MLN64 and N- 62 StAR to transport cholesterol between artificial membranes and to its inner membrane site of use in placental mitochondria. Urea denaturation experiments show that N- 218 MLN64 undergoes a pH-dependent and denaturant-dependent structural transition to a molten globule state, as reported previously for N-62 StAR. N-218 MLN64 stimulated cholesterol transfer between artificial phospholipid vesicles with an initial rate of 6.5 mol/min.mol N-218 MLN64. Both N-218 MLN64 and N-62 StAR stimulated cholesterol transfer to the inner mitochondrial membrane, as evidenced by a 6-fold stimulation of pregnenolone synthesis with saturating transporter. This stimulation was seen only after the endogenous cholesterol in the steroidogenic pool of the isolated mitochondria was first depleted. No stimulation was observed by N-218 MLN64 or N-62 StAR when 20alpha-hydroxycholesterol was added as substrate for P450scc, confirming that these proteins stimulate P450scc activity by enhancing cholesterol transport. MLN64 levels in placental JEG-3 cells were unresponsive to stimulation by 8-bromo-cAMP over 24 h. These data show that human N-218 MLN64 and N-62 StAR have similar biophysical and functional properties and are able to stimulate steroidogenesis in a human placental system, which normally lacks StAR. The results reveal that with saturating MLN64, steroidogenesis by placental mitochondria proceeds at near-maximal rate.