Objective: Though determining the free hexafluoro-isopropanol (HFIP) concentration in blood when SD rats had been anesthetized with different sevoflurane concentrations for different time, to observe the change of free HFIP concentration in rats’ blood, and analyze the relationship between inhaled sevoflurane concentration and free HFIP concentration in blood among the three groups. Methods: One hundred and fourteen healthy adult SD rats were selected, in which 6 rats were used to determine the blood/gas partition coefficient (B/G) of HFIP, and the rest 108 rats were randomly divided into three groups (S1, S2 and S3) with 36 rats in each group, and anesthetized by sevoflurane with different concentrations (1.16%, 2.32% and 3.48%), respectively. Every 6 rats in each group were accepted anesthesia by inhaled sevoflurane with a preset concentration for 0.5 1.0, 1.5, 2.0, 3.0 and 4.0 h, respectively. First, the blood/gas partition coefficient of HFIP samples was measured by the two-stage headspace equilibration, and then the actual free HFIP concentration in the blood of anesthetized rats was measured by the one-stage headspace equilibration. At the same time, the sevoflurane concentration and blood/gas partition coefficient in the blood of anesthetized rats were determined by the two-stage headspace equilibrium method. Both the curves of free HFIP concentration in blood and inhaled sevoflurane concentration vs. anesthesia time were made, and the relationship among them was analyzed. Results: Free HFIP can be detected and its peak value occurred at 1.0 hr after inhaled sevoflurane anesthesia in each group, and after that decreased along with the prolong of anesthesia time. Compared with group S1, the free HFIP concentration in group S2 was higher at each corresponding time point (P < 0.05), but except for the time point of 4.0 h, no significant differences were found in group S3 at corresponding time points (P > 0.05). Compared with group S2, the free HFIP concentration in group S3 was significantly lower at each corresponding time point (P < 0.05). Conclusion: The free HFIP concentration in blood showed a tendency from initial increase to subsequent decrease with the prolongation of sevoflurane anesthesia time. When the inhaled sevoflurane concentration increased to 3.48%, the generation of free HFIP was decreased and the metabolism of sevoflurane was inhibited.