Persistent firing is believed to be a crucial mechanism for memory function including working memory. Recent in vivo and in vitro findings suggest an involvement of metabotropic glutamate receptors (mGluRs) in persistent firing. Using whole-cell patch-recording techniques in a rat entorhinal cortex (EC) slice preparation, we tested whether EC layer III neurons display persistent firing due to mGluR activation, independently of cholinergic activation. Stimulation of the angular bundle drove persistent firing in 90% of the cells in the absence of a cholinergic agonist. The persistent firing was typically stable for > 4.5 min at which point persistent firing was terminated by the experimenter. The average frequency of the persistent firing was 2.1 Hz, ranging from 0.4 to 5.5 Hz. This persistent firing was observed even in the presence of atropine (2 μm), suggesting that the persistent firing can occur independent of cholinergic activation. Furthermore, ionotropic glutamate and GABAergic synaptic blockers (2 mm kynurenic acid, 100 μm picrotoxin and 1 μm CGP55845) did not block the persistent firing. On the other hand, blockers of group I mGluRs (100 μm LY367385 and 20 μm MPEP) completely blocked or suppressed the persistent firing. An agonist of group I mGluRs (20 μm DHPG) greatly enhanced the persistent firing induced by current injection. These results indicate that persistent firing can be driven through group I mGluRs in entorhinal layer III neurons, suggesting that glutamatergic synaptic input alone could enable postsynaptic neurons to hold input signals in the form of persistent firing.