Activation of the vertebrate locomotor network is mediated via glutamatergic synaptic drive, normally initiated by the brainstem. Previous investigations have studied the role of glutamate receptors, especially NMDA receptors, in generating and regulating locomotor pattern generation. Few studies, however, have focused on the role of AMPA receptors in shaping network activity, especially with regard to their rapid desensitization. It is important to determine if AMPA receptor desensitization plays a role in regulating neuronal network activity. We examined this question on both the network and synaptic levels in the lamprey (Lampetra fluviatilis) spinal cord using a selective and potent inhibitor of AMPA receptor desensitization, cyclothiazide (CTZ). The solvent dimethyl sulfoxide (DMSO) is commonly used to dissolve this drug, as well as many others. Unexpectedly, the vehicle alone already at 0.02%, but not at 0.01%, caused significant increases in EPSP amplitudes and NMDA-induced locomotor frequency. The results indicate that DMSO may have a profound influence when used at or above 0.02%, a concentration 10-50 times less than those most commonly used. Subsequently we applied CTZ at or below 10 µM concentrations (DMSO 0.01%). CTZ (1.25-5 µM) caused a marked and significant increase in EPSPs mediated by non-NMDA receptors and in both AMPA- and NMDA-induced locomotor frequency, but no effects on EPSPs mediated by NMDA receptors. From the effects of CTZ it is apparent that AMPA receptor desensitization plays an important role in determining locomotor frequency and that this is likely due to its limiting function on AMPA receptor mediated EPSPs.