Na,K,2Cl co-transporter (NKCC2), the primary NaCl uptake pathway in the thick ascending limb of Henle, is expressed in three different full-length splice variants, called NKCC2F, NKCC2A, and NKCC2B. These variants, derived by differential splicing of the variable exon 4, show a distinct distribution pattern along the loop of Henle, but the functional significance of this organization is unclear. By introduction of premature stop codons into exon 4B, specific for the B isoform, mice with an exclusive NKCC2B deficiency were generated. Relative expression levels and distribution patterns of NKCC2A and NKCC2F were not altered in the NKCC2B-deficient mice. NKCC2B-deficient mice did not display a salt-losing phenotype; basal plasma renin and aldosterone levels were not different from those of wild-type mice. Ambient urine osmolarities, however, were slightly but significantly reduced. Distal Cl concentration was significantly elevated and loop of Henle Cl absorption was reduced in microperfused superficial loops of Henle of NKCC2B-deficient mice. Because of the presence of NKCC2A in the macula densa, maximum tubuloglomerular feedback responses were normal, but tubuloglomerular feedback function curves were right-shifted, indicating reduced sensitivity in the subnormal flow range. Plasma renin concentration in NKCC2B-deficient mice was reduced under conditions of salt loading compared with that in wild-type mice. This study shows the feasibility of generating mice with specific deletions of single splice variants. The mild phenotype of mice that are deficient in the B isoform of NKCC2 indicates a limited role for NKCC2B for overall salt retrieval. Nevertheless, the high-affinity NKCC2B contributes to salt absorption and macula densa function in the low NaCl concentration range.