Vitamin D is essential for health and wellbeing. Latest studies show that the lack of this vital secosteroid is associated with development of many pathological conditions. For that reason the vitamin D research has been very active during recent years. The purpose of this study was to determine the 1,25-dihydroxyvitamin D concentrations of human serum samples by using the IDS-iSYS 1,25-Dihydroxy Vitamin D kit and IDS-iSYS immunochemical automated analyzer. The assigner for this study was biotechnology company ValiRx Finland Oy. The assay was performed in co-operation with University of Oulu, Department of Clinical Chemistry. The methodological basis for this study was comparative. The scheme was to repeat the assay that was already once accomplished by ValiRX Finland Oy, and compare the renewed test results with the former results. The reliability of the first test results were questioned, for the analysis was carried out with a preliminary test kit. In this study we used parallel samples from the earlier study and a test kit from an updated lot. The main objective was to determine the possible reliance and the differences on the results between the two analysis in average. Our intension was also to survey the main sources of error in the test procedure, and contemplate the reasons for the possible deviations. Based on the statistical analysis, differences between the previous and the replicated results exist. The determined differences of the means can be considered statistically significant. Linear dependence between compared assays is noticeable, but not as strong as assumed. During the test procedure was discovered that the pretreatment process was particularly susceptible to faults. The samples were provided ready by the assigner, so the authors of the study did not have all the exact knowledge of the preanalytical factors that might have had influence on the results.