7-Dehydrocholesterol (7-DHC) is the most oxidizable lipid molecule reported to date, with a propagation rate constant for free radical peroxidation that is 200 times that of cholesterol. To better understand the high reactivity of 7-DHC and elucidate the reaction mechanism, we synthesized conjugated and skipped nonconjugated cholestadienols that would give one of the two putative pentadienyl-radical intermediates formed in 7-DHC peroxidation. The additional dienols include 6,8(9)-dienol, 5,8(14)-dienol, 6,8(14)-dienol, and the biologically important 8-dehydrocholesterol (8-DHC; 5,8(9)-dienol). We found that all of the dienols are significantly (at least 40 times) more reactive than cholesterol. Among them, dienols leading to the formation of the pentadienyl radical in ring B (termed endo-B) of the sterol are more reactive than those leading to the pentadienyl radical spanning rings B and C (termed exo-B). By comparing the oxysterol profile formed from 7-DHC and those formed from 8-DHC and 5,8(14)-dienol, products formed from abstraction of the hydrogen atoms at C-9 and C-14 (H-9 or H-14 mechanism) were clearly differentiated. When the oxidation was carried out in the presence of the good hydrogen atom donor α-tocopherol, the oxysterol profile of 7-DHC peroxidation differed distinctly from the profile observed in the absence of the antioxidant and resembles more closely the profile observed in biological systems. This study suggests that oxidative stress and the accumulation of oxysterols should be considered as two key factors in cholesterol biosynthesis or metabolism disorders, where dienyl sterol intermediates are accumulated.