Phospholipase C1 (PLC1) exists as two splice variants, PLC1a (150 kDa) and PLC1b (140 kDa), which differ only in their C-terminal sequences of 64 and 31 amino acids, respectively. The 3 C-terminal amino acid residues of PLC1a comprise a PDZ-interacting domain, whereas the PLC1b sequence has no PDZ-interacting domain but contains unique proline-rich domain 5 residues from the C terminus. PLC1a is localized in the cytoplasm, whereas PLC1b targets to the sarcolemma and is enriched in caveolae. Deletion of 3 amino acids from the C terminus of PLC1b did not alter its sarcolemmal localization, but deletion of the entire unique 31 amino acid sequence caused cytosolic localization. A myristoylated 10 amino acid peptide from the C terminus of PLC1b selectively dissociated N-terminally GFP-tagged PLC1b from the sarcolemma and inhibited PLC responses to ₁-adrenergic agonists, with a half maximal effective concentration of 12 ± 1.6 µM (mean±SE, n=3). A similar peptide from PLC1a was without effect at concentrations below 100 µM. Thus, the extreme C-terminal sequences of the PLC1 splice variants determine localization and, thus, function. In cardiomyocytes, responses initiated by ₁-adrenergic receptor activation involve only PLC1b, and the selective targeting of this splice variant to the sarcolemma provides a potential therapeutic target to reduce hypertrophy, apoptosis, and arrhythmias.—Grubb, D. R., Vasilevski, O., Huynh, H., and Woodcock, E. A. The extreme C-terminal region of phospholipase C1 determines subcellular localization and function; the "b" splice variant mediates ₁-adrenergic receptor responses in cardiomyocytes.