SummaryBackground25 (OH) vitamin D3 (25(OH)D) and parathyroid hormone (PTH) are important regulators of calcium homeostasis. The aim of this study was to retrospectively determine the cut–off for sufficient 25(OH)D in a four-season region and the influence of age, seasons, and gender on serum 25(OH)D and PTH levels.MethodsLaboratory results of 9890 female and 2723 male individuals aged 38.8±22.1 years who had simultaneous measurements of 25(OH)D and PTH were retrospectively analyzed by statistical softwares. Serum 25(OH)D and PTH levels were measured by a mass spectrometry method and by an electrochemiluminescence immunoassay, respectively.ResultsMean serum 25(OH)D levels showed a sinusoidal fluctuation throughout the year and were significantly (p<0.01) higher in summer and autumn. On the other hand, PTH levels were significantly higher (p<0.01) in women and showed an opposite response to seasonal effects relative to 25(OH)D. Lowest levels of 25(OH)D were detected in people aged between 20 and 40 years whereas PTH hormone levels were gradually increasing in response to aging. The significant exponential inverse relationship that was found between PTH and 25(OH)D (PTH=exp(4.12–0.064*sqrt(25(OH)D)) (r=–0.325, R– squared=0.105, p<0.001)) suggested that the cut–off for sufficient 25(OH)D should be 75 nmol/L.ConclusionsOur retrospective study based on large data set supports the suitability of the currently accepted clinical cut–off of 75 nmol/L for sufficient 25(OH)D. However, the issue of assessing Vitamin D deficiency remains difficult due to seasonal variations in serum 25(OH)D. Therefore, PTH measurements should complement 25(OH)D results for diagnosing Vitamin D deficiency. It is imperative that seasonally different criteria should be considered in future.