In the present study, we investigated the role of orexinergic systems in the activation of midbrain dopamine neurons. In an in vitro study, exposure to either orexin A or orexin B under superfusion conditions produced a transient increase in the intracellular Ca(2+) concentration through the phospholipase C (PLC)/protein kinase C (PKC) pathway via G(q11)alpha or Gbetagamma subunits in midbrain cultured neurons, which were shown to be tyrosine hydroxylase (TH)-positive cells, but not in purified midbrain astrocytes. Here we show that in vivo injection with a selective PKC inhibitor chelerythrine chloride or 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-1-methyl-1H-indol-3-ylmaleimide HCl (Ro-32-0432) into the ventral tegmental area (VTA) significantly suppressed the place preference and increased levels of dopamine in the nucleus accumbens (NAcc) induced by intra-VTA injection of orexins. These results strongly support the idea that activation of the orexin-containing neuron in the VTA leads to the direct activation of mesolimbic dopamine neurons through the activation of the PLC/PKC pathway via G(q11)alpha or Gbetagamma-subunit activation, which could be associated with the development of its rewarding effect.