A cholinomimetic substance was isolated from desiccated venom of Dendroaspis polylepis by one-dimensional ascending paper chromatography. The migratory and staining properties of the substance were compared with those of standard acetylcholine. Pharmacological and biochemical identification was carried out on various in vitro and in vivo biological test objects as well as with high-voltage paper electrophoresis. The assay of the cholinomimetic substance was done on both superfused guinea pig ileum and hamster stomach strips. The cholinomimetic substance content was 2.44 - 3.46mg/0.96gm of total protein in the desiccated venom.