The effect of palytoxin on the transport of ascorbic acid into cultured bovine adrenal chromaffin cells was examined by measuring the accumulation of radiolabeled ascorbic acid within cells. Ascorbic acid transport into these cells was inhibited by palytoxin in a concentration-dependent manner, and this inhibitory action of palytoxin was shown to be noncompetitive and irreversible. Neither the Na+/K+-pump activity in the intact cells nor the Na+,K+-adenosine 5'-triphosphatase activity in the plasma membranes was significantly influenced by this toxin at concentrations inhibiting ascorbic acid transport. In contrast to the effect of palytoxin on ascorbic acid transport, glucose transport into these cells was not significantly affected by this toxin. These findings indicate that palytoxin can inhibit ascorbic acid transport into adrenal chromaffin cells without affecting Na+,K+-adenosine 5'-triphosphatase activity in the plasma membranes. Furthermore, because palytoxin discriminated between ascorbic acid transport and glucose transport, the data provide new evidence that the transport of ascorbic acid and that of glucose may be mediated by different mechanisms in the adrenal medullary cell.