Only L- and N-type high voltage-activated calcium currents (HVA ICa) have been demonstrated in identified embryonic spinal motoneurons. However, pharmacological experiments suggest that other HVA ICa, including P-type, govern neurotransmitter release at the adult neuromuscular junction. We sought to analyse if embryonic motoneurons express these other ICa, using the whole-cell voltage-clamp method on motoneurons purified by a new metrizamide-panning technique from E15 rat embryos. In addition to L-type dihydropyridine-sensitive and N-type omega-GVIA-sensitive currents, motoneurons express two other HVA ICa. One has properties related to the P-type channel currents described in Purkinje cells: it is inhibited by the peptide omega-agatoxin-IVA with a maximal effect at 100-200 nM. The inhibited current has a characteristic sustained component during depolarizing test pulses. Furthermore, 50-100 nM concentrations of omega-agatoxin-IVA reduce the increase in cytoplasmic calcium concentration observed after depolarization. The other HVA ICa is resistant to saturating concentrations of verapamil, omega-conotoxin GVIA and omega-agatoxin-IVA which block L, N and P-type HVA ICa, respectively. These results suggest that it is now possible to dissect, using a simple method of purification, the properties of the ICa in embryonic mammalian motoneurons and to provide pharmacological evidence for multiple calcium channels which may be involved in regulation of their activity during development.