We describe the cloning and characterization of the first human members, hKv9.1 and hKv9.3, of the electrically silent delayed-rectifying-like K+ channel subfamily. Their modulatory effects on the electrically active subfamily member hKv2.1 are also quantified. The hKv9 K+ channels were isolated from a human lens epithelium cDNA library, but both hKv9.1 mRNA and hKv9.3 mRNA were found to coexist with the mRNA for hKv2.1 in a large number of human tissues. The hKv9.1 gene is composed of a minimum of five exons, with at least two alternatively spliced exons in the 5'-untranslated region (UTR). In contrast, the hKv9.3 gene is intronless across the coding region, 3'-UTR, and all of the analyzed 5'-UTR. Radiation hybrid mapping localized the hKv9.1 gene to 20q12 and the hKv9.3 gene to 2p24. Each electrically silent subunit, when coexpressed with hKv2.l, slows deactivation and inactivation compared with hKv2. 1 expressed alone. In addition, each results in an increment in the single channel conductance.