We have studied factors affecting biological variation in total plasma alkaline phosphatase in a population of 32 329 apparently healthy subjects four years old or older. Quantification of the bone and liver isoenzymes after thermal denaturation made it possible to specify the contributions of each isoenzyme to variations in the total activities. The main factors that modify plasma alkaline phosphatase activity are age, sex, hormonal state (puberty or menopause), and morphometric parameters (height, body weight, or degree of overweight). The bone isoenzyme is mainly responsible for the variations associated with age, sex, and puberty and to some extent with the menopause. Activity of the liver isoenzyme was also altered at the menopause and by certain drugs, such as oral contraceptives and blood-lipid-lowering agents. These data allow us to propose reference limits for total plasma, bone, and liver alkaline phosphatases according to age and sex.