Stimulation of the greater occipital nerve produces excitation of second order neurons in the trigeminocervical complex. Given that neck pain is very common in primary headache disorders, this convergent excitation may play a role in pain referral from cervical structures. While previous studies have demonstrated a physiological model for this convergence, this study sought an anatomical approach to examine the distribution of second order neurons in the trigeminocervical complex receiving greater occipital nerve input. In addition, the role of glutamatergic NMDA receptor activation within the trigeminocervical complex in response to cervical afferents was studied. Noxious stimulation of the occipital muscle in rat using mustard oil and mineral oil produced significantly altered Fos expression in the trigeminocervical complex compared with the surgical control (H(4)=31.3, P<0.001, Kruskal-Wallis). Baseline expression was 11 (median, range 4, 17) fos positive cells in the trigeminocervical complex, occipital muscle treated with mustard oil produced 23 (17, 33) and mineral oil a smaller effect of 19 (15, 25) fos positive cells, respectively (P=0.046). The effects of both mustard and mineral oil were reversed by the NMDA-receptor antagonist MK801. This study introduces a model for examining trigeminocervical complex activity after occipital afferent stimulation in the rat that has good anatomical resolution and demonstrates involvement of glutamatergic NMDA receptors at this important synapse.