The content of vitamin D in pork produced in conventional systems depends on the vitamin D concentration in the pig feed. Both vitamin D-3 and 25-hydroxyvitamin D-3 (25(OH)D-3) are essential sources of dietary vitamin D; however, bioavailability assessed by serum 25(OH)D-3 concentration is reported to be different between the two sources. Furthermore, the relationship between serum 25(OH)D-3 level and the tissue content of vitamin D-3 and 25(OH)D-3 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 D-3 and 25(OH)D-3 in the pig feed for 49 d before slaughter. Concurrently, the 25(OH)D-3 level in serum was investigated as a biomarker to assess the content of vitamin D-3 and 25(OH)D-3 in pig tissues. Adipose tissue, white and red muscle, the liver and serum were sampled from pigs fed feed containing either vitamin D-3 or 25(OH)D-3 at 5, 20, 35 or 50 mu g/kg feed for 7 weeks before slaughter. The tissue 25(OH)D-3 level was significantly higher in the pigs fed 25(OH)D-3 compared with those fed vitamin D-3, while the tissue vitamin D-3 level was higher in the pigs fed vitamin D-3 compared with those fed 25(OH)D-3. The content of 25(OH)D-3 in the different tissues fully correlated with the serum 25(OH)D-3 level, whereas the correlation between the tissue content of vitamin D-3 and serum 25(OH)D-3 was dependent on the source of the ingested vitamin D-3.