A novel method for detecting the membrane receptors of Substance P (SP) has been developed. The method does not require radioactive derivatives of SP and is based on quantitation of specifically bound biotinylated SP (Bt-SP), the analysis being performed after destruction of the ligand-receptor complex in acidic conditions and Bt-SP extraction into solution. The acidic extract from the Bt-SP-membrane complex after neutralization is added to immulon microELISA plate coated with affinity purified anti-SP-antibodies. The sensitivity of subsequent detection of Bt-SP enhances by using a new type of the streptavidin conjugate with a polymeric form of horseradish peroxidase. Enzyme immunoassay conditions were optimized using [125I]-labelled SP derivative. The developed method allows the determination of femtomoles of SP in a sample and has a sensitivity comparable to that of radioligand analysis.