Sensitization of dorsal horn neurons following injury may underlie the generation of secondary hyperalgesia and so the chemical basis of sensitization is now receiving considerable attention. The present study used microiontophoretic applications of excitatory amino acids (EAA's) and substance P (SP) to test their roles in the sensitization of primate spinothalamic tract (STT) neurons. Of 70 STT cells examined in laminae I-VI of the dorsal horn, 40 showed an increase in responses to one or more EAA's following their co-application with SP. The increased responses were usually specific to either N-methyl-D-aspartate (NMDA) or to the non-NMDA agonists, quisqualate (QUIS) or D,L-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA). The enhancement of EAA responses was long-lasting (> 15 min) in 18 cases, was accompanied by similarly long-lasting increases in responses to mechanical stimulation of the receptive field in 14 cases and was accompanied by an increase in responses to either glutamate (Glu) or aspartate (Asp) in eleven cases. A global decrease in all EAA responses tested was produced in 26 other STT neurons. The inhibition, unlike the increases, was generalized to both NMDA and non-NMDA ligands, was long-lasting in only six cases and was never accompanied by a change in the responses to mechanical stimuli. The excitatory and inhibitory effects of SP on the responses to NMDA were uniformly reversed by the NK-1 receptor selective antagonist, CP96345. In contrast, only the inhibitory effects of SP on the responses to QUIS or AMPA were reversed by CP96345. The long-lasting enhancement of EAA responses by SP may follow the combined synaptic release of the natural ligands in vivo, resulting in the sensitization of dorsal horn neurons and secondary hyperalgesia. However, the reductions in EAA responses produced by SP are problematic for this hypothesis and need further elucidation.