The early kinetics of the conversion of cholesterol (A) to (22R)-22-hydroxycholesterol (B), (20R,22R)-20,22-dihydroxycholesterol (C) and pregnenolone (D) has been studied with bovine adrenocortical mitochondrial aceton-dried powder preparations. The sequential appearance of B, C, and D was demonstrated. During the lag period of D appearance, B, and C approached steady state levels, at which time the formation of D approximated linearity. The initial rate of B appearance approximated the rate of the linear phase of pregnenolone formation. When cholesterol was initially incubated in an 18O2-enriched atmosphere, the gas phase abruptly changed to air and incubation continued for a relatively short period, there was a drop in the 18O content of the recovered B and C. These results demonstrated for the first time the turnover of these compounds as they formed in the system from cholesterol, without the use of exogenously added tracer B or C. The 18O content of the recovered glycol was lower at position C-20 than at C-22, as would be expected from a consecutive process involving an initial oxygen attack of cholesterol at C-22. These results suggest the sequence A leads to B leads to C leads to D as the basic mechanism for the conversion of cholesterol to pregnenolone.