Ca2+ enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca2+ entry through activation of pertussis toxin-sensitive G proteins leading to activation of K+ channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca 2+ channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three α1 (α1A, α 1C, and α1D), four β, and one α 2-δ subunit in rat pituitary GH3 cells; α 1B and α1S transcripts were absent. GH3 cells express multiple alternatively spliced α1D mRNAs. Many of the α1D transcript variants encode "short" α1D (α1D-S) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other α 1 subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (α1D-L). We cloned and expressed the predominant α1D-S transcript variants in rat brain and GH3 cells and their α1D-L counterpart in GH3 cells. Unlike α1A channels, α1D channels exhibited current-voltage relationships similar to those of native GH3 cell Ca2+ channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced α1D transcripts form functional Ca2+ channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of α1D-S subunit, is not sufficient to confer sensitivity to inhibitory G proteins.