The Ste20 kinases SPAK and OSR1 regulate Na+ transport in the distal nephron. Western blot analysis of kidney lysates revealed the presence of multiple SPAK fragments in the kidney medulla, which are thought to have inhibitory effects on OSR1 in the thick ascending limb of Henle [1, 2]. The nature of these fragments is still not completely understood. Based on a 49 kDa band size found in multiple tissues, we proposed earlier that translation also starts at an alternative methionine, resulting in a shorter protein sometimes called SPAK2 [3]. Analysis of Expressed Tag Sequences databases also revealed the presence of a shorter SPAK transcript consistent with a 34 kDa protein (KS-SPAK) [2]. Here, we demonstrate using a C-terminal anti-SPAK antibody that mouse kidney lysate cleaves in a dose- and time-dependent manner a fusion protein consisting of GST followed by full-length SPAK. The incubation results in five to seven proteolytic fragments with two of the fragments having sizes compatible with SPAK2 and KS-SPAK. We further confirm that thrombin, which protease site is located at the end of GST, produces only one fragment of a larger molecular size. When we used equal protein amounts from lysates prepared from mouse brain, spleen, liver, and kidney, we only observed the cleavage pattern with kidney, indicating that the protease(s) is/are enriched in this tissue. We demonstrate that the cleavage is resistant to aprotinin (2.5 μM), leupeptin (40 μM), PMSF (3 mM), and EDTA (5 mM), indicating inefficacy of traditional protease inhibitors. The cleavage was also resistant to pepstatin (1-10 μM), and CuSO4 (1 mM) or doxycycline (200 μM), potentially eliminating cathepsins and metalloproteases as possible proteolytic enzymes. We show, however, that the cleavage was sensitive to DTT, as proteolysis was significantly reduced with 1 mM of the reducing agent and completely eliminated at 10 mM. This observation indicates that the protease is stabilized by di-sulfide bridges. The protein cleavage occurred over a wide temperature range (4oC – 45oC), but was impaired at 55oC and eliminated at 65oC and higher temperatures. Finally, we tested SPAK cleavage over a wide pH range (pH 5.5 – 9.5) and made the following observation: the proteolysis leading to the higher size fragments occurred equally over the entire pH range, whereas the proteolysis leading to the smaller two fragments was significantly inhibited by basic pH and markedly activated by acidic pH. This observation indicates the possible involvement of two distinct proteases. Future work will seek to identify the amino acid sequences in SPAK targeted for proteolytic cleavage and determine the identity of the protease(s) involved.