The most important enzyme in hepatic drug metabolism is cytochrome P450 3A4. Published in vitro data indicate that vitamin D may up-regulate the expression of the CYP3A4 gene. Individual vitamin D levels are highly dependent on sunlight exposure and show great seasonal variability in northern countries. The aim of the present study was to investigate whether plasma concentrations of CYP3A4 drug substrates exhibit seasonal changes compatible with a stimulatory effect of vitamin D on drug metabolism. Three immunosuppressants (tacrolimus, sirolimus, and cyclosporine) were analyzed, because these CYP3A4 drug substrates are subject to long-term use and repeated concentration determinations. In addition, mycophenolic acid was included in the analysis as a control drug independent of CYP3A4 metabolism. Concentration-to-dose ratios were extracted from the Karolinska Therapeutic Drug Monitoring database and compared between the 3-month periods of lowest and highest vitamin D levels. Sirolimus and tacrolimus levels showed seasonal variability that was highly consistent with changes in vitamin D; for example, significantly lower drug concentrations in July to September than in January to March. As expected, no significant difference was evident for mycophenolic acid, but this result was also the case with cyclosporine, possibly due to cross-reactivity of CYP3A4-mediated metabolites with the immunoassay used for quantification. In conclusion, there is cyclic variation in blood levels of important immunosuppressants throughout the year that correlates with UV light-dependent changes in vitamin D levels. Even though a causal relationship remains to be established, it is suggested that individual differences in vitamin D may contribute to variability in drug metabolism and disposition.