Normal activity of brain and plasma cholinesterase in higher vertebrates is known to be affected by age, genetics, circadian rhythms, sex, endocrine function, and reproductive status. Various stressors (e.g., nutritional plane, ambient temperature, disease) have also been demonstrated to alter cholinesterase activity in birds and mammals. Naturally-occurring and stress-induced variation in cholinesterase activity should be considered when the biomarker is used to assess exposure to organophosphorus and carbamate pesticides. In addition to pesticide metabolism (activation and detoxication) and physiological condition, basal cholinesterase activity may also be a determinant of sensitivity to cholinesterase-inhibiting pesticides. The present review examines the biological and stress-induced sources of variability in brain and blood cholinesterase activity of non-exposed vertebrates, and the toxicological and hazard assessment ramifications of such variability.