The enzyme cytochrome P450 11A1 cleaves the C20-C22 carboncarbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carboncarbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO3+). Compound I can either (i) abstract an OH hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carboncarbon bond cleavage step was tested using O-18-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen (O-18(2)), and coupled assays were used to trap the labile O-18 atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the O-18 content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an O-18 atom into either of its carboncarbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.