The chapter provides an overview and historical perspective of the many available methods for cholinesterase (ChE) activity determination. Due to the almost universal use of the spectrophotometric assay in wildlife toxicology, the remainder of the chapter focuses on this technique. Sources of variability in the assay are discussed, including assay temperature, instrumentation, degree of solubilization of membrane-bound enzyme, and lipid concentration in serum samples. Methods to control these sources of error are offered. Consideration is given to methods of differentiating the activity of the various esterases (for example, acetylcholinesterase and butyrylcholinesterase). A brief review is provided of commercial kits available for field and/or laboratory testing of samples for ChE activity. The chapter includes a discussion of future innovations and refinements of the ChE assay, including the use of reactivation techniques and adaptation of the assay for use with 96-well microtitration plates. (Copyright (c) 1991 Elsevier Science Publishers B.V.)