Block of N-type Ca channels by omega-conotoxin GVIA (CgTx) was studied in freshly dissociated bullfrog and rat sympathetic neurons. With 2–5 mM Ba as charge carrier, CgTx blocked almost all of the high-threshold Ca channel current recorded in the presence of nimodipine (3 microM) to block L-type Ca channels. Toxin block reversed slowly (time constant approximately 1 hr) in frog cells and even more slowly in rat cells. CgTx block was faster and more potent in rat cells than frog cells. The rate of block was proportional to CgTx concentration, consistent with 1:1 binding of CgTx to channels. When the external Ba concentration was increased, the development of block was slower, consistent with competition between CgTx and Ba for a binding site. The recovery from block was somewhat faster in higher external Ba. Some cells had significant current remaining in saturating concentrations of nimodipine and CgTx, especially with high Ba concentrations in the external solution. The current resistant to nimodipine and CgTx was activated at lower depolarizations than the CgTx-sensitive current and had faster activation and inactivation kinetics, but unlike low- threshold T-type current, the resistant current had rapidly decaying tail currents.