Mutations that disrupt a PY motif in epithelial Na⁺channel (ENaC) subunits increase surface expression of Na⁺channels in the collecting duct, resulting in greater Na⁺reabsorption. Recently, Nedd4 and Nedd4–2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4–2 (hNedd4–2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5′-exons were identified by 5′-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3′-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4–2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4–2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na⁺transport regulation. In vitro binding studies demonstrated that individual PY motifs of α-, β-, and γ-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4–2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4–2 in the regulation of ENaC and define protein domains important for Nedd4–2 function.