A bovine homologue of the rat and human epithelial Na+ channel subunits, α-rENaC and α-hENaC, was cloned. The cDNA clone, termed α-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed α-bENaC- associated Na+ current was not responsive to elevations in adenosine 3',5'- cyclic monophosphate but could be stimulated by phorbol 12-myristate 13- acatate, an activator of protein kinase C. α-bENaC also formed amiloride- sensitive chimeric channels when coexpressed with the rat β- and γ-ENaC subunits in Xenopus oocytes. α-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.