Auxiliary (beta) subunits of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the gating properties of the functional channel complex. Here we show that an avian beta1-subunit also stimulates the trafficking of BK(Ca) channels to the plasma membrane in HEK293T cells and in a native population of developing vertebrate neurons. One C-terminal variant of BK(Ca) alpha-subunits, called the VEDEC isoform after its five last residues, is largely retained in intracellular compartments when it is heterologously expressed in HEK293T cells. A closely related splice variant, called QEERL, shows high levels of constitutive trafficking to the plasma membrane. Co-expression of beta1-subunits with the VEDEC isoform resulted in a large increase in surface BK(Ca) channels as assessed by cell-surface biotinylation assays, whole cell recordings of membrane current, and confocal microscopy in HEK293T cells. Co-expression of beta1-subunits slowed the gating kinetics of BK(Ca) channels, as reported previously. Consistent with this, overexpression of beta1-subunits in a native cell type that expresses intracellular VEDEC channels, embryonic day 9 chick ciliary ganglion neurons, resulted in a significant increase in macroscopic Ca(2+)-activated K(+) current. Both the cytoplasmic N- and C-terminal domains of avian beta1 are able to bind directly to VEDEC and QEERL channels. However, overexpression of the N-terminal domain by itself is sufficient to stimulate trafficking of VEDEC channels to the plasma membrane, whereas overexpression of either the cytoplasmic C-terminal domain or the extracellular loop domain did not affect surface expression of VEDEC.