Human biomonitoring has become a primary tool for chemical exposure characterization in a wide variety of contexts: population monitoring and characterization at a national level, assessment and description of cohort exposures, and individual exposure assessments in the context of epidemiological research into potential adverse health effects of chemical exposures. The accurate use of biomonitoring as an exposure characterization tool requires understanding of factors, apart from external exposure level, that influence variation in biomarker concentrations. This review provides an overview of factors that might influence inter- and intraindividual variation in biomarker concentrations apart from external exposure magnitude. These factors include characteristics of the specific chemical of interest, characteristics of the likely route(s) and frequency of exposure, and physiological characteristics of the biomonitoring matrix (typically, blood or urine). Intraindividual variation in biomarker concentrations may be markedly affected by the relationship between the elimination half-life and the intervals between exposure events, as well as by variation in characteristics of the biomonitored media such as blood lipid content or urinary flow rate. Variation across individuals may occur due to differences in time of sampling relative to exposure events, physiological differences influencing urinary flow or creatinine excretion rates or blood characteristics, and interindividual differences in metabolic rate or other factors influencing the absorption or excretion rate of a compound. Awareness of these factors can assist researchers in improving the design and interpretation of biomonitoring studies.