Background: Primary hyperparathyroidism is often diagnosed by high calcium levels in blood. It is well known that calcium levels are dependent on vitamin D and Parathyroid hormone (PTH). Since vitamin D has a seasonal variation the calcium levels might also be influenced by seasonal variation. If a seasonal variation in calcium levels exists, this must be considered in the investigation of suspected hyperparathyroidism. The aim of the present study was to investigate the possible influence and magnitude of the seasonal variation of vitamin D and PTH on calcium levels. Method: In the present study the individual seasonal variation of 25-hydroxyvitamin D [25(OH) D], PTH and calcium in 69 healthy volunteers living at latitudes with extremely variable seasonal exposure to sunlight have been investigated. Results: As expected the 25(OH) D levels were significantly higher (42%) in summer compared to winter. PTH levels were significantly lower (7%) in summer than in winter. The mean serum concentration of calcium was 1% higher in August than in February, however not statistically significant. A good agreement between summer and winter calcium values was confirmed by Bland-Altman analysis. Conclusion: This study did not show any clinically important influence of seasonal variation of 25(OH) D and PTH on calcium that may influence a clinician's decision to investigate suspected hyperparathyroidism.