The metabolites of dl-3-n-butylphthalide(NBP), a novel drug with promising protective action against cerebral ischemia, was studied in rats. Two main in vitro metabolites of NBP, M I and M II, were isolated and purified from rat liver microsome incubating system by using HPLC. The structure elucidation was mainly accomplished by spectral studies(UV, 1H-NMR, MS). Within 24 h following i.g. 3H-NBP, the total radioactivity excreted in urine and feces was 73.7% of the dose. Comparing with previous study, within 72 h following i.g. NBP, the total prototype drug excreted in urine and feces was 2.53% of the dose. This result excludes the possibility that NBP accumulates in vivo. The urine and brain homogenate of the rats(i.g. 3H-NBP) were analyzed by TLC. M I and M II were found in urine and M I was found in brain only. Furthermore, the ratio of radioactive M I to proptype drug was 1:1 in rat brain within 1 h following i.g. 3H-NBP. So, M I and M II were supposed to be the two main in vivo metabolites of NBP and M I might be an active metabolite.