With K-band EPR (Electron Paramagnetic Resonance), ENDOR (Electron Nuclear-Double Resonance), and EIE (ENDOR-induced EPR) techniques, three free radicals (RI-RIII) in L-lysine hydrochloride dihydrate single crystals X-irradiated at 298 K were detected at 298 K, and six radicals (R1, R1', R2-R5) were detected if the temperature was lowered to 66 K from 298 K. R1 and RI dominated the central portion of the EPR at 66 and 298 K, respectively, and were identified as main chain deamination radicals, -OOC(C) over dotH(CH2)(4)(NH3)(+). R1' was identified as a main chain deamination radical with the different configuration from RI at 66 K, and it probably formed during cooling the temperature from 298 to 66 K. The configurations of RI, R1', and RI were analyzed with their coupling tensors. R2 and R3 each contain one alpha- and four beta-proton couplings and have very similar Ems at three crystallographic axes. The two-layer ONIOM calculations (at B3LYP/6-31G(d,p):PM3) support that R2 and R3 are from different radicals: dehydrogenation at C4, -OOCCH(NH3)+CH2(C) over dotH(CH2)(2)(NH3)(+), and dehydrogenation at CS, -OOCCH(NH3)(+)(CH2)(2)(C) over dotHCH(2)(NH3)(+), respectively. The comparisons of the coupling tensors indicated that R2 (66 K) is the same radical as RII (298 K), and R3 is the same as RIII. Thus, RII and RIII also are the radicals of C4 and C5 dehydrogenation. R4 and R5 are minority radicals and were observed only when temperature was lowered to 66 K. R4 and R5 were only tentatively assigned as the side chain deamination radical, -OOCCH (NH3)(+)(CH2)(3)(C) over dotH(2), and the radical dehydrogenation at C3, -OOCCH-(NH3)(+)(C) over dotH(CH2)(3)(NH3)(+), respectively, although the evidence was indirect. From simulation of the EPR (B//a, 66 K), the concentrations of RI, R1', and R2-R5 were estimated as: R1, 50%; R1', 11%; R2, 14%; R3, 16%; R4, 6%; R5, 3%.