Human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA clones were isolated from an HL-60 cell cDNA library by using a reverse transcription/polymerase chain reaction-generated human cDNA probe. The 24-hydroxylase cDNA consists of a 1539-bp open reading frame encoding a 513-amino acid polypeptide. Protein sequence analysis shows that the human 24-hydroxylase is 90% homologous (82% identical) to that of the rat, with 100% homology in the 21-amino acid heme-binding region. Northern blot analysis showed that the 24-hydroxylase cDNA probe hybridized to a 3.4-kb mRNA species. Treatment of HL-60 cells with 0.1 microM 1,25-dihydroxyvitamin D3 for 24 hr produced a 30-fold increase in the 24-hydroxylase mRNA level. This result is consistent with previous studies in the same cell line, in which 24-hydroxylase activity was elevated to a maximum in 24 hr by a similar treatment with 1,25-dihydroxyvitamin D3. To verify the identity of these isolated cDNA clones, two polymerase chain reaction-amplified human 24-hydroxylase cDNA fragments containing the entire coding region were used to produce 24-hydroxylase enzyme activity in two genetic expression systems. Transient levels of 24-hydroxylase activity were measured in transfected mammalian COS-1 cells and in recombinant baculovirus-infected Spodoptera frugiperda (Sf21) insect cells.