The effects of trypsin, bile extract, temperature and acid-based condition for the in vitro excystation of Haplorchis taichui metacercariae were studied. At 37°C, approximately half the number of metacercariae excysted when exposed to 1% trypsin for 15 minutes with no more excystation found beyond this time. Increasing trypsin concentration seemed to reduce the excystation rate while bile extract was, however, unlikely to be an absolute requirement. A temperature of 37°-41°C yielded a similar excystation result in combination with 1% trypsin; however, less excystation occurred at a lower temperature of 35°C. The acid-based environment of pH 8 gave the best excystation result in association with 1% trypsin at a temperature of 39°C. Higher and lower basicity produced a smaller excystation rate. An environmental condition of 1% trypsin at pH 8 and a temperature of between 37°-41°C was recommended for the in vitro excystation of H. taichui metacercariae. The relatively broad temperature and pH range condition for the excystation of H. taichui corresponded with various definitive hosts that were infected naturally by this fluke.