Biochemical markers of bone turnover have been shown to provide valuable information for the diagnosis and monitoring of metabolic bone disease. However, these dynamic indexes are influenced by a number of factors that need to be clearly identified to improve their clinical usefulness. To evaluate the contributions of anthropometric, life style, and environmental variables on bone turnover, biochemical markers of bone metabolism were determined in a population-based sample of 580 adults, aged 50-81 yr (297 men and 283 women). Subjects were recruited during 14 consecutive months within the framework of the European Vertebral Osteoporosis Study. Serum total and bone-specific alkaline phosphatase (S-BAP), serum C-terminal propeptide of type I collagen, and serum osteocalcin (S-OC) were measured as bone formation markers. Urinary total pyridinoline and deoxypyridinoline were included as bone resorption indexes. In females, serum levels of 25-hydroxyvitamin D3 were significantly higher (P < 0.01) in summer (May-September) than in winter (October-April), whereas no significant differences were found in males. In both sexes, no seasonal changes were seen in serum PTH. In males, serum total alkaline phosphatase (P < 0.01), S-BAP (P < 0.001), and S-OC (P < 0.05) were significantly higher in winter than in summer. During the same period, females had higher values of S-BAP (P < 0.05), S-OC (P < 0.01), and urinary pyridinoline and deoxypyridinoline (P < 0.001, respectively). Univariate analyses of the effects of life style habits on markers of bone metabolism revealed that in females, regular alcohol consumption and current smoking led to a suppression of markers of bone turnover, whereas in males, only alcohol intake was associated with such changes. In contrast, physical activity was associated with higher levels of bone formation markers and reduced levels of bone resorption indexes in both sexes. As shown by multivariate regression analyses, seasonal variations accounted for more of the variability in most biomarkers (up to 12%) than any of the other anthropometric or life style factors except age. This effect may be attributed to subclinical vitamin D deficiency during the winter period, which is common in countries of the northern hemisphere. We conclude that seasonal variation contributes significantly to the biological variability of bone turnover and needs consideration when interpreting the results of bone marker measurements.