Unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons of the granular layer. Previous studies have shown that there are two distinct subsets of UBCs present in the mice cerebellar cortex: calcium-binding protein calretinin (CR) positive and metabotropic glutamate receptor (mGluR)1alpha positive. In this study, we identify phospholipase C (PLC) beta4 as an antigenic marker of a novel subset of UBCs. Double immunolabeling reveals that none of the CR+ subset expresses PLCbeta4. In contrast, most members of the mGluR1alpha subset also express PLCbeta4. In addition, 65% of the PLCbeta4+ subset does not express mGluR1alpha. Thus, there are three distinct UBC subsets in the mouse cerebellum: CR+/PLCbeta4-/mGluR1alpha-, PLCbeta4+/mGluR1alpha-/CR-, and mGluR1alpha+/PLCbeta4+/CR-. Each has a different topographical distribution, both between lobules and mediolaterally within the vermis. The development of PLCbeta4 expression in UBCs is exclusively postnatal--first seen only at P12 and mature at about 3 weeks. A distinct subset of PLCbeta4+ UBCs is also present in primary cerebellar cultures.