Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5–20 mm Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed ≈ 20% L-, ≈ 45% N- and ≈ 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20–200 nmω-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nm in 5 mm Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 μmω-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the α1E channel. The current activates around −20 mV in 20 mm Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP- and ω-CTx-GVIA, and displays anomalous sensitivity to ω-Aga-IVA and ω-CTx-MVIIC (Hong & Chang (1995) J. Physiol. (Lond.), 482, 283–290; Lin & Lin-Shiau (1997) Eur. J. Neurosci., 9, 817–823].