RT-PCR analysis showed elevated expression of 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase) and of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) in well differentiated human colon carcinomas in comparison to normal mucosa. Further tumor progression is associated with a rise in 1α-OHase but with no significant change in 24-OHase mRNA expression. Accordingly, HPLC analysis of 25-hydroxy-vitamin D 3 metabolism in freshly isolated tumor cells indicated that well to moderately differentiated colon cancers in situ are able to produce 1α,25-dihydroxyvitamin D 3 (1α,25-(OH) 2 D 3 ) and convert it through 24-OHase activity into side-chain modified metabolites, 1,24,25-(OH) 3 -D 3 and 1,25-(OH) 2 - 24-oxo-D 3 . Likewise, 25-(OH)-D 3 is metabolized into 24,25-(OH) 2 D 3 , 23,25-(OH) 2 D 3 , and 23,25-(OH) 2 -24-oxo-D 3 . Poorly-differentiated cancers expressed low levels of 1α-OHase mRNA, whereas 24-OHase was even over-expressed. RT-PCR and HPLC analysis of vitamin D metabolism in primary culture cell clones strongly suggested that the extent of endogenously produced 1α,25-(OH) 2 -D 3 was inversely related to 24-OHase activity, which could thus limit the antimitotic efficacy of 1α,25-(OH) 2 -D 3 particularly at late stages of colon cancer progression.