We describe here (1) the heterogeneous expression of Ca2+-independent transient (A-type) K+ channel alpha-subunits (Kv1.4, Kv3.3, Kv3.4, Kv4.2 and Kv4.3) in rat smooth muscle, heart and brain, (2) the molecular cloning and tissue distribution of a novel alternatively spliced variant of an A-type K+ channel alpha-subunit, Kv4.3, and (3) the functional expression of A-type K+ channels in HEK293 cells by the transfection with the novel splice variant of Kv4.3. A cDNA encoding this splice variant was identified from rat vas deferens by RT-PCR cloning. This cDNA clone contains a 1965 bp open reading frame that encodes for a protein of 655 amino acids. It has a 19 amino acid insertion in comparison with Kv4.3 previously reported in rat brain. RT-PCR analyses showed that the mRNAs of this longer variant are abundantly expressed in a number of smooth muscles of the rat, and that the mRNAs of the previously reported clones are absent. The longer splice variant is very weakly expressed in brain, but is the major product in heart.