The stimulation of C-fiber sensory neurons is known to induce activation of the ERK MAP kinase signaling pathway in the spinal cord dorsal horn. In this study we have elucidated some of the signaling components of C-fiber transmission responsible for ERK activation. Using an in vitro slice preparation of the mouse spinal cord dorsal horn, we compared the release of substance P (SP) and BDNF with the activation of ERK in postsynaptic neurons. We observed that primary afferent stimulation recruiting C-fibers was required for both SP and BDNF release and ERK activation in post-synaptic dorsal horn neurons. Glutamate transmission via NMDA and mGluR1 but not AMPA receptors was critical to this ERK activation. BDNF signaling via TrkB receptors but not SP signaling via NK(1) were also involved in ERK recruitment. In conclusion, glutamate and BDNF are the important C-fiber signaling components for ERK activation in dorsal horn neurons.