Summary The effects of substance P (SP) on the isolated pupillary sphincter of the cat and rabbit were studied. The sphincter was incubated in an organ bath which was filled with Krebs solution saturated with 100% O 2 and kept at 36 ± 0.5 °C. The contractile and relaxant responses were recorded with an isotonic transducer. In rabbits, exogenously applied SP showed a potent contractile effect, and ED 50 value was 1.3 × 10 −9 M. However, in cats, SP produced little or no response at a concentration of 5 × 10 −6 M. Electrical field stimulation of the rabbit pupillary sphincter produced a muscle contraction composed of rapid and slow components. It was confirmed that the first component was cholinergic and the second component SP-ergic. By contrast, electrical field stimulation of the cat pupillary sphincter produced a rapid contraction followed by a slow relaxation. The rapid contractile component was accelerated by pretreatment with 10 −6 M physostigmine and reduced by 10 −6 M atropine, while the slow relaxant component was not affected by 10 −6 M physostigmine, 10 −6 M atropine 10 −6 M phentolamine or 10 −6 M propranolol. A releaser and depleter of SP, capsaicin (10 −5 M), produced a marked relaxant response in the cat pupillary sphincter. After desensitization to capsaicin, the slow relaxant component disappeared in response to electrical field stimulation.