The beta subunits of voltage-dependent calcium channels bind the pore-forming alpha(1) subunit and play an important role in the regulation of calcium channel function. Recently, we have identified a new splice variant of the beta(4) subunit, which we have termed the beta(4d) subunit. The beta(4d) subunit is a truncated splice variant of the beta(4b) subunit and lacks parts of the guanylate kinase (GK) domain and the C-terminus. The calcium current in BHK cells expressing alpha(1C) and alpha(2)delta with the beta(4d) subunit was as small as that without the beta(4d) subunit. Western blot analysis revealed that beta(4d) protein was expressed to a lesser extent that the beta(4b) protein. In addition, a GST pull down assay showed that the beta(4d) subunit could not interact with the alpha(1) subunit of the calcium channel. Collectively, our results suggest that the GK domain of the beta subunit is essential for the expression of the functional calcium channel.