P-type ATPases require both alpha- and beta-subunits for functional activity. Although an alpha-subunit for colonic apical membrane H-K-ATPase (HKcalpha) has been identified and studied, its beta-subunit has not been identified. We cloned putative beta-subunit rat colonic H-K-ATPase (HKcbeta) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat beta-subunits. Northern blot analysis demonstrates that this HKcbeta is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcbeta mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase beta1 mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKcbeta mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcbeta fusion protein established expression of a 45-kDa HKcbeta protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcbeta protein expression only in apical membranes. Physical association between HKcbeta and HKcalpha proteins was demonstrated by Western blot analysis performed with HKcbeta antibody on immunoprecipitate of apical membranes of rat distal colon and HKcalpha antibody. Tissue-specific upregulation of this beta-subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKcalpha protein provide compelling evidence that HKcbeta is the putative beta-subunit of colonic H-K-ATPase.