Nitric oxide (NO) is pivotal to bone physiology. In the central nervous system constitutive, Ca2+-calmodulin regulated NO synthase activity and glutamate signalling are intimately linked. Since L-glutamate signalling occurs in bone and is implicated in bone regulation, we have investigated the effect of L-glutamate on NO synthase in bone-derived cells. Treatment of marrow stromal cells with L-glutamate reduced basal NO synthase activity by 40%. Imaging showed that L-glutamate caused a rapid, usually localised and slowly-reversible fall in [Ca2+]i. This effect was resistant to disruption of intracellular Ca2+ stores but sensitive to extracellular La3+ or omission of extracellular Ca2+, demonstrating that glutamate acts by inhibition of membrane Ca2+ influx. The only previous description of such an effect of L-glutamate is via activation of the group III receptor, mGluR6, in the retina. Using Western blotting and RT-PCR we detected mGluR6 protein and transcripts in marrow stromal cells. The effects of L-glutamate on NOS activity and [Ca2+]i in marrow stromal cells were abolished by a group III mGluR inhibitor, (S)-2-amino-2-methyl-4-phosphonobutyric acid. Recording of membrane potential showed that, similarly to the effects of retinal mGluR6 activation, L-glutamate induced membrane hyperpolarisation (−16 ± 2 mV), which was also sensitive to group III mGluR inhibition. L-glutamate had no effect on cAMP levels. We conclude that activation of a group III mGluR in bone marrow stromal cells inhibits a Ca2+-permeable plasma membrane channel, reducing [Ca2+]i and suppressing generation of NO. These observations directly link bone L-glutamate signalling to processes central to bone growth and regulation. © 2005 Wiley-Liss, Inc.