We have previously reported an association of 14-3-3 epsilon isoform with calmodulin. Using the voltage-clamp technique, the present study investigated the potential role of 14-3-3 in modulating the Ca2+-activated Cl- channel (CaCC) endogenously expressed in Xenopus oocytes. Injection of 14-3-3 epsilon antisense oligodeoxynucleotides resulted in potentiation of the ionomycin-induced Cl- current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppressed the antisense-potentiated current. The data suggest that 14-3-3 epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin-dependent pathway. The potential role of 14-3-3 epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed.