Increasing evidence suggests glutamate has an important modulatory role in mechanosensory endings in the peripheral nervous system1 in addition to its well-established role in the central nervous system. Annulospiral stretch-sensitive nerve endings in skeletal muscle – muscle spindles – express a novel glutamate receptor with an atypical pharmacological profile most closely matching the phospholipase D (PLD)-coupled glutamate receptor1. Although reported in the hippocampus by a number of groups2,3, the receptor has never been isolated or sequenced. We previously reported spindle expression of a ~102 kDa probable mGluR5 splice variant and an unidentified ~110 kDa protein capable of binding our unique biotinylated ligand – ZCZ-1804. Here we report the use of spindles extracted from the rat deep masseter muscle – an enriched source of nerve endings5 – to further investigate and isolate the novel glutamate system. Adult male Sprague Dawley rats (weight 200 – 450g) were euthanised by CO2 overdose (ASPA 1986, 63/2010/EU) and deep masseters removed. For 1D gels and affinity chromatography, spindles were extracted as previously described4. For immunohistochemistry, muscles were fixed overnight in 4% formaldehyde, teased then squashed and incubated in primary antibody for 48 hr followed by secondary antibody for 1 hr. Hydrophobic spindle protein was separated on 1D gels. Bands were selected by molecular weight and analysed by mass spectrometry. A band at ~100 kDa did not match any known proteins on the Mascot database. However, a subset of unassigned peptides showed strong sequence homology with glutamate receptors following NCBI-based sequence alignment, suggesting sufficiently representativeprotein isolation. Functional clustering analysis usingIPA (Ingeuinty Pathway Analysis) software identified PLD as a potential regulatoryhub, suggesting the protein is likely to signal through PLD. Protein isolation was cross-validated by affinity chromatography. Eluate had immunoreactivity to mGluR5 antibody at ~102 kDa confirming successful isolation of one protein by two separate strategies. Immunohistochemistry showed the mGluR5-like protein was not localised to the annulospiral nerve endings, but in nociceptive fibres parallel to spindles, suggesting the mGluR5-like protein is not involved in mechanosensation. An mGluR5 variant was isolated by two separate strategies. Although we initially suspected the expression of a novel heterodimer in spindles, the mGluR5-like protein is not localised to the mechanosensory portion of spindles, suggesting it is not involved in the stretch response. Future work will focus on localising and sequencing the ZCZ-binding protein in our quest to isolate the receptor protein.