The content of vitamin D in pork produced in conventional systems depends on the vitamin D concentration in the pig feed. Both vitamin D₃ and 25-hydroxyvitamin D₃ (25(OH)D₃) are essential sources of dietary vitamin D; however, bioavailability assessed by serum 25(OH)D₃ concentration is reported to be different between the two sources. Furthermore, the relationship between serum 25(OH)D₃ level and the tissue content of vitamin D₃ and 25(OH)D₃ is unknown. The objective of this study was to investigate the potential of increasing the content of vitamin D in different pig tissues by increasing the levels of vitamin D₃ and 25(OH)D₃ in the pig feed for 49 d before slaughter. Concurrently, the 25(OH)D₃ level in serum was investigated as a biomarker to assess the content of vitamin D₃ and 25(OH)D₃ in pig tissues. Adipose tissue, white and red muscle, the liver and serum were sampled from pigs fed feed containing either vitamin D₃ or 25(OH)D₃ at 5, 20, 35 or 50 µg/kg feed for 7 weeks before slaughter. The tissue 25(OH)D₃ level was significantly higher in the pigs fed 25(OH)D₃ compared with those fed vitamin D₃, while the tissue vitamin D₃ level was higher in the pigs fed vitamin D₃ compared with those fed 25(OH)D₃. The content of 25(OH)D₃ in the different tissues fully correlated with the serum 25(OH)D₃ level, whereas the correlation between the tissue content of vitamin D₃ and serum 25(OH)D₃ was dependent on the source of the ingested vitamin D₃.