Analytical, within-subject and between-subject components of variation have been determined for total alkaline phosphatase (ALP) and its bone isoform, osteocalcin, and tartrate-resistant acid phosphatase (TR-ACP) in serum specimens from a cohort of 10 healthy subjects over a 1-month period. From these data, we have calculated the desirable analytical imprecisions, the indices of individuality, the critical differences for significant change detection, and the number of specimens required to estimate the homeostatic setpoint of an individual. For total ALP and its bone isoform, most of the observed variability was biologic, whereas osteocalcin and TR-ACP also had relatively high analytical variability. Total and bone ALP were also the parameters showing the lowest within-subject variability, whereas TR-ACP showed the lowest interindividual fluctuation. Osteocalcin and bone ALP had marked individuality, showing that the use of population-based reference limits is inadequate for their interpretation. The applicable differences required for two results to be significantly different (p < or = 0.05) are bone ALP: 20%; osteocalcin: 29%; and TR-ACP: 35%. The results demonstrate that the biochemical markers of bone turnover studied are of limited use to screen for metabolic bone disorders, but can be useful adjuncts for monitoring groups of patients in longitudinal studies.