A sensitive and specific double-antibody enzyme immunoassay (EIA) for a substance P (SP)-like immunoreactive substance (SP-IS) was developed. For competitive reactions, the SP-antibody was incubated with SP standard (or sample) and β-D-galactosidase labeled Tyr⁸-SP (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 10fmol/well of SP. Using the present EIA, SP-ISs in human saliva and plasma were determined. The level of SP-IS in human saliva was about 7pmol/l, which was almost three times higher than that in human plasma.