Recent studies indicated that junctophilins (JPs) contribute to the formation of junctional membrane structures in excitable cells by interacting with the plasma membrane and spanning the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane. In the brain, functional crosstalk between cell-surface and intracellular channels is proposed in the "subsurface cistern" as the junctional membrane complex observed in neurons. So far, three JPs have been identified as tissue-specific subtypes derived from different genes; JP-1 is specifically expressed in skeletal muscle, JP-2 is detected throughout muscle cell types, and JP-3 is predominantly expressed in the brain. In this paper, we report a novel JP subtype, JP-4, encoded in the human (chromosome 14q11.1) and mouse (chromosome 14C1-2) genomes. Cloning the cDNA showed that JP-4 shares characteristic structural features with other JP subtypes, and Northern and Western blot analyses demonstrated its brain-specific expression. In situ hybridization analysis revealed that both JP-3 and JP-4 mRNAs are expressed in discrete neuronal sites, and their overall regional distribution patterns were similar in the brain. Furthermore, both the JP mRNAs and subsurface cistern showed somatodendritic localization in hippocampal pyramidal neurons. The results obtained suggest the collaborative contribution of JP-3 and JP-4 to the subsurface cistern formation in neurons.