An amiloride-sensitive Na+ channel is found in the apical plasma membrane of high resistance, Na+ transporting epithelia. We have developed a method for the identification of this channel based on the use of a new high affinity photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), and anti-amiloride antibodies to identify photolabeled polypeptides. NMBA specifically labels the putative Na+ channel in bovine kidney microsomes. A 130-kDa polypeptide is detected on immunoblots with anti-amiloride antibodies. NMBA is a potent inhibitor of Na+ transport in the established amphibian kidney epithelial cell line A6, and specifically labels a 130-kDa polypeptide. We utilized both NMBA photolabeling and [3H]benzamil binding in order to examine the cellular pool of putative channels following hormonal regulation of Na+ transport. This pool is not significantly altered by the mineralocorticoid agonist aldosterone or antagonist spironolactone, despite a 3.8-fold difference in transepithelial Na+ transport.