Persistent elevation of blood pressure (BP), termed hypertension (HTN), affects one in three adults in the US and more than one billion individuals worldwide. A genome-wide association study (GWAS) by our group identified intronic sequence variants that show association to systolic BP (SBP) in the gene STK39. The protein encoded by STK39, SPAK, plays a critical role in the maintenance of vascular volume, and physiologic and genetic evidence solidifies the significance of SPAK in BP regulation. Therefore, it is important to identify and characterize all functional variants in the STK39 gene. We sequenced exons and noncoding conserved elements in STK39 in a subset of Amish Family Diabetes Study (AFDS) participants and identified four coding variants. In exon 1, there are three insertion/deletion (in/del) polymorphisms that alter the length of a relatively-uncharacterized proline-alanine rich domain. In exon 11, the nonsynonymous SNP rs56031549 creates a putative novel phosphorylation site by replacing an alanine with a threonine in the regulatory domain of SPAK. These four variants have minor allele frequencies ranging from 0.02 to 0.28 in European Americans (EA). In the AFDS, the 21- and 3-nucleotide in/dels show association to SBP under an additive model (p < 0.05). We also detected suggestive evidence of association between SBP and the 3-nucleotide deletion in another group of Amish subjects (additive model, p = 0.07). Analysis of these variants in the outbred EA population of the GenNet study showed a trend towards significance for rs56031549 to SBP (p = 0.07). Functional characterization based on an in vitro kinase assay revealed that the STK39 haplotype of the 21-nucleotide deletion and 6-nucleotide deletion (9 amino acid deletion) resulted in greater kinase activity that is not attributable to the phosphorylation status of SPAK's active site. Thus, the association between the 21-nucleotide deletion and BP might lead to a variant SPAK protein with greater kinase activity that increases renal sodium reabsorption by cotransporters such as NCC and NKCC2. This finding suggests that pharmaceutical manipulation of SPAK activity may be a novel and effective approach for the treatment of HTN.