Abstract: We report the existence, in Torpedo marmorata tissues, of a cholinesterase species (sensitive to 10−5M eserine) that differs from acetylcholinesterase (AChE, EC 188.8.131.52) in several respects: (a) The enzyme hydrolyzes butyrylthiocholine (BuSCh) at about 30% of the rate at which it hydrolyzes acetylthiocholine (AcSCh), whereas Torpedo AChE does not show any activity on BuSCh. (b) It is not inhibited by 10−5M BW 284C51, but rapidly inactivated by 10−8M diisopropyl-fluorophosphonate. (c) It does not exhibit inhibition by excess substrate up to 5 × 10−3M AcSCh. (d) It does not cross-react with anti-AChE antibodies raised against purified Torpedo AChE. This enzyme is obviously homologous to the “nonspecific” or pseudocholinesterase (pseudo-ChE, EC 184.108.40.206) that exists in other species, although it is closer to “true” AChE than classic pseudo-ChE in several respects. Thus, it shows the highest Vmax with acetyl-, and not propionyl- or butyrylthiocholine, and it is not specifically sensitive to ethopropazine. Pseudo-ChE is apparently absent from the electric organs, but represents the only cholinesterase species in the heart ventricle. Pseudo-ChE and AChE coexist in the spinal cord and in blood plasma, where they contribute to AcSCh hydrolysis in comparable proportions. Pseudo- ChE exists in several molecular forms, including collagen-tailed forms, which can be considered as homologous to those of AChE. In the heart the major component of pseudo-ChE appears to be a soluble monomeric form (G1). This form is inactivated by Triton X-100 within days. In addition, it is converted, on storage of the extracts, into more rapidly sedimenting, Triton X-100-resistant forms (G2 and G4). A G4 form exists in spinal cord and plasma. Collagen-tailed forms, A12 and A8, were characterized in high-salt extracts from spinal cord and heart ventricle. Pseudo-ChE molecular forms sediment slightly slower than the corresponding AChE forms. The two polymorphic enzyme systems, AChE and pseudo-ChE, therefore arose as early as elasmobranchs during the evolution of vertebrates.