Background: Multiple sclerosis is a disorder with genetic and environmental contributions. Factors of relevance include the CYP27B1 gene and low serum 25(OH)D levels. The interplay with 1,25(OH)2D, the active form of vitamin D, is not well understood. Objective: We aim to evaluate (i) the association between 25(OH)D & 1,25(OH)2D, (ii) factors predicting 1,25(OH)2D after 25(OHD) levels are taken into account, (iii) the interplay between vitamin D metabolism genes and vitamin D metabolites for FCD risk. Methods: This investigation utilised data from the Early Environment and Type 1 Diabetes Prevention Project; (EET1DPP; 241 cases, 322 controls), the Southern Tasmanian MS Longitudinal study (MSL; 198 prevalent MS cases), and the Ausimmune Study (279 FCD cases, 577 controls). Serum vitamin D metabolites, SNP data and other factors were examined. A genetic function score (GFS) was constructed using key vitamin D metabolism genes. A variety of statistical approaches were used. Results: Correlation between 25(OH)D and 1,25(OH)2D was not high (r=0.11-0.58 across samples). In some samples, accounting for Vitamin D-related genes and their interaction with 25(OH)D levels improved the prediction of 1,25(OH)2D from 25(OH)D alone. Vitamin D-related SNPs were associated with FCD risk, either directly or exhibiting interaction with 1,25(OH)2D levels. The CYP27B1 and GC gene variants were associated with FCD, with an interaction between the GC variant and 1,25(OH)2D levels. Conclusion: Incorporating genetic information may improve 1,25(OH)2D levels prediction beyond 25(OH)D levels alone in some settings. The demonstrated interplay between these vitamin D metabolites and related genes requires replication.