Pharmacologically distinct calcium channels have been characterized in dissociated cutaneous sensory neurons and motoneurons of the larval lamprey spinal cord. To enable cell identification, sensory dorsal cells and motoneurons were selectively labeled with fluorescein-coupled dextran amine in the intact spinal cord in vitro before dissociation. Calcium channels present in sensory dorsal cells, motoneurons, and other spinal cord neurons were characterized with the use of whole cell voltage-clamp recordings and specific calcium channel agonist and antagonists. The results show that a transient low-voltage-activated (LVA) calcium current was present in a proportion of sensory dorsal cells but not in motoneurons, whereas high-voltage-activated (HVA) calcium currents were seen in all neurons recorded. The different components of HVA current were dissected pharmacologically and similar results were obtained for both dorsal cells and motoneurons. The N-type calcium channel antagonist omega-conotoxin-GVIA (omega-CgTx) blocked >70% of the HVA current. A large part of the omega-CgTx block was reversed after washout of the toxin. The L-type calcium channel antagonist nimodipine blocked approximately 15% of the total HVA current. The dihydropyridine agonist (+/-)-BayK 8644 markedly increased the amplitude of the calcium channel current. The BayK-potentiated current was not affected by omega-CgTx, indicating that the reversibility of the omega-CgTx effect is not due to a blockade of L-type channels. Simultaneous application of omega-CgTx and nimodipine left approximately 15% of the HVA calcium channel current, a small part of which was blocked by the P/Q-type channel antagonist omega-agatoxin-IVA. In the presence of the three antagonists, the persistent residual current (approximately 10%) was completely blocked by cadmium. Our results provide evidence for the existence of HVA calcium channels of the N, L, and P/Q types and other HVA calcium channels in lamprey sensory neurons and motoneurons. In addition, certain types of neurons express LVA calcium channels.