Voltage-gated potassium channel beta subunits are cytoplasmic proteins that co-purify with the pore-forming alpha subunits. One of these subunits, Kv beta 1 from rat brain, was previously demonstrated to increase the rate of inactivation of Kv1.1 and Kv1.4 when co-expressed in Xenopus oocytes. We have cloned and characterized a novel voltage-gated K+ channel beta subunit. The cDNA, designated Kv beta 3, has a 408-amino acid open reading frame. It possesses a unique 79-amino acid N-terminal leader, but is identical with rat Kv beta 1 over the 329 C-terminal amino acids. The Kv beta 3 transcript was found in many tissues, but was most abundant in aorta and left ventricle of the heart. Co-expression of Kv beta 3 with K+ channel alpha subunits shows that this beta subunit can increase the rate of inactivation from 4- to 7-fold in a Kv1.4 or Shaker B channel. Kv beta 3 had no effect on Kv1.1, unlike Kv beta 1 which can increase rate of inactivation of this alpha subunit more than 100-fold. Other kinetic parameters were unaffected. This study shows that voltage-gated K+ channel beta subunits are present outside the central nervous system, and that at least one member of this family selectively modulates inactivation of K+ channel alpha subunits.