The thermoisomerization of previtamin D3 to vitamin D3 is the last step in the synthesis of vitamin D3 in human skin. Kinetic and thermodynamic studies of this reaction in human skin and an organic solvent revealed that not only the equilibrium of the reaction was shifted in favor of vitamin D3 formation in human skin (equilibrium constant K at 37 degrees C = 11.44) compared to hexane (K = 6.15), but also the rate of the reaction was increased by more than 10-fold in human skin (T1/2 at 37 degrees C = 2.5 h) when compared to hexane (T1/2 = 30 30 h). This extraordinarily fast reaction rate was also confirmed in vitro in chicken skin and in vivo in human subjects. The enthalpy change for the reaction determined by the van't Hoff plot was delta H degree = -21.58 kJ mol-1 in human skin and delta H degree = -15.60 kJ mol-1 in hexane. Arrhenius plots showed that the activation energies for both the forward and the reverse reactions were lower in human skin (Ea1 = 71.05 kJ mol-1 and Ea2 = 92.63 kJ mol-1) than in hexane (Ea1 = 84.90 kJ mol-1 and Ea2 = 100.5 kJ mol-1). Activation parameters for the reaction in human skin and in hexane were also reported. Subcellular fractionation of human epidermal tissue revealed that most epidermal 7-dehydrocholesterol and previtamin D3 were in the membrane fraction, while only 20% were in the cytosol. The interaction of previtamin D3 with intracellular lipids and/or proteins in skin may be responsible for the increased vitamin D3 formation rate in the skin.