1. The purpose of the present study was to investigate whether there was a cooperative interaction between substance P (SP) and glutamate (GLU) administered subcutaneously on Adelta and C primary afferent fibre activity in dorsal hairy skin of the rat in vivo. The single unit activities of Adelta and C afferent fibres were recorded by isolation of fibre filaments from the dorsal cutaneous nerve branches and the effects of subcutaneous injections of low doses of SP, GLU and SP + GLU on activity were determined. 2. Sub-threshold doses of SP (1 micro mol/L, 10 microL) administered subcutaneously into the dorsal hairy skin had no effect on the afferent discharges of either Adelta or C units. 3. The afferent discharges of 35% (11/31) of Adelta fibres and 33% (6/18) of C fibres were increased by local injection of the submaximal doses of GLU (10 micro mol/L, 10 microL) into the receptive fields. 4. The GLU-induced excitatory response was significantly enhanced by coinjection of subthreshold doses of SP. The mean discharge rates of Adelta fibres and C fibres were increased from 5.84 +/- 1.54 and 5.02 +/- 2.65 impulses/min to 19.91 +/- 4.35 and 17.58 +/- 5.59 impulses/min, respectively, whereas the excitatory proportions of Adelta and C fibres were increased from 35 and 33% to 84 and 83%, respectively. The duration of the excitation for Adelta fibres and C fibres was also significantly increased after coinjection of SP + GLU compared with that observed when either substance was given alone. 5. The present study provides electrophysiological evidence for an interaction between receptors for SP and GLU on the fine fibres activities in rat hairy skin, which may be involved in the mechanisms of hyperalgesia.