CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2D3 using rat CYP24A1. Both 1,25(OH)2D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid-1) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid-1, with 24-oxo-1,23,25- trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3D3 displaying the highest kcat (34 min -1) and 24-oxo-1,23,25(OH)3D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3D3 occurs with the highest catalytic efficiency (kcat/Km) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as an accumulation of intermediates, indicating that they dissociate after each catalytic step. © 2014 FEBS.