Tissue transglutaminase (tTG) is a multifunctional enzyme with transglutaminase crosslinking (TGase), GTP binding, and hydrolysis activities that play a role in many different disorders. We identified, characterized, and investigated the function and stability of two alternatively spliced forms of tTG using biochemical, cellular, and molecular biological approaches. Using a human aortic vascular smooth muscle cells (VSMC) cDNA library, we identified two cDNAs encoding C-terminal truncated forms, tTG_V1 and tTG_V2. tTG_V1,2 mRNAs were synthesized by a rare splicing event using alternate splice sites within exons 12 and 13 of the tTG gene, respectively. Quantitative PCR and immunoblotting demonstrated that there was unique expression and localization of tTG_V1,2 compared with tTG in human umbilical vein endothelial cells (HUVECs), VSMC, and leukocytes. The loss of C-terminal 52 amino acid residues (AAs) in tTG_V1,2 altered GTP binding, enhanced GTP hydrolysis, rendered the variants insensitive to GTP inhibition, and resulted in <10% residual Ca⁺²-dependent TGase activity. Transfection in HEK293 demonstrated a 28- and 5-fold reduction in the expression of tTG_V1 and tTG_V2, respectively, demonstrating that the C-terminal GTP-binding domain is important in stabilizing and promoting the half-life of tTG. The altered affinity for GTP allowed tTG_V1,2 to exhibit enhanced TGase activity when there is a transient increase in Ca⁺² levels. The abundance of tTG_V1,₂ and its distinct intracellular expression patterns in human vascular cells and leukocytes indicate these isoforms likely have unique physiological functions.--Lai, T. S., Liu, Y., Li, W., Greenberg, C. S. Identification of two GTP-independent alternatively spliced forms of tissue transglutaminase in human leukocytes, vascular smooth muscle and endothelial cells.