Four substance P (SP) analogues were tested on reaction time (RT) in the tail flick test and on the decrease in RT produced by the SP homologue physalaemin. The four analogues were [D-Trp7,9,10]SP, denoted A, [D-Pro4,D-Trp7,9,Nle11]SP-(4-11), denoted B, [D-Pro2,D-Trp7,9,10]SP, denoted C and [D-Pro4,D-Trp7,9,10,Phe11]SP-(4-11), denoted D. Physalaemin alone (1.89 nmol) reduced RT. The analogue A, at 3.25 nmol, blocked the effects of physalaemin without altering basal RT. The analogues B and D, which block the action of physalaemin in peripheral tissues, had neither agonistic nor antagonistic effects in doses up to 6.5 nmol. The replacement of L-Pro2 by D-Pro2 in the analogue A yielded the analogue C, which had no antagonistic activity. All analogues produced a flaccid paralysis of the hindlimbs and the tail; this effect was inconsistent, though, occurring only in some rats, and appearing in some cases after the first administration of the analogue yet in other cases only after a subsequent administration. Because B and D are inactive in the spinal cord, our results suggest that physalaemin activates receptors in the spinal cord different from those it activates in peripheral tissues. Furthermore, because all four analogues produced a flaccid paralysis none is suitable for use as an SP antagonist in vivo in the CNS.