Well-resolved ESR spectra of free pentadienyl radicals have been observed under photoirradiation of di-tert-butylperoxide (ButOOBut) and polyunsaturated fatty acids in the absence of O2, allowing us to determine the hfc values. The hfc values of linoleyl radical indicate that the spin density is the largest at the C-11 position. The linoleyl radical is readily trapped by O2 to produce the peroxyl radical (11-HPO •) in which O2 is added mainly at the C-11 position of the pentadienyl radical as indicated by the comparison of the ESR spectra of peroxyl radicals derived from linoleic acid and [11,11-2H 2]linoleic acid. The peroxyl radical (13-HPO•), which is initially formed by the hydrogen abstraction from 13-(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13-HPOD) by ButO•, is found to isomerize to 11-HPO• via removal of O2 from 13-HPO• and addition of O2 to linoleyl radical to produce 11-HPO•. This finding supports an idea of O2 entering via a specific protein channel, which determines the stereo- and regiochemistry of the biradical combination between O2 and linoleyl radical in lipoxygenases.