Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in H 2 O than in D 2 O, while the second carbon‑carbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both H 2 O and D 2 O. We also studied the temperature-dependent decay of the peroxo-ferric and hydroperoxo-ferric intermediates generated by cryoradiolysis of the corresponding oxygenated heme proteins at 77 K. For both pregnenolone and 17-hydroxypregnenolone, annealing of the peroxo-intermediates was observed at lower temperatures in H 2 O than in D 2 O. In contrast, no solvent isotope effect was detected when progesterone or 17-hydroxyprogesterone were used as substrates. These differences are attributed to their different positioning in the P450 active site with respect to the heme bound peroxo (Fe-OO - ) moiety, which is in agreement with earlier structural and spectroscopic investigations. Analysis of the samples run in both H 2 O and in D 2 O, where 17-hydroxyprogesterone is the substrate, demonstrated significant (∼25%) yield of androstenedione product relative to the oxygenated starting material.