In vitro excystation studies were carried out on the metacercariae cysts of Paragonimus heterotremus obtained from naturally infected crabs Potamon spp. The effects of elastase, trypsin, trypsin-dog bile, trypsin-bile salt, and dithiothreitol (DTT) were examined. The trypsin-dog bile medium stimulated maximum excystation. Of the media that contained 1 mM DTT, the optimum conditions for the excystation were shown to be pH 9, temperature of 39–40 C, and osmolarity of 250–350 mOsm. The DTT acceleration was antagonized by all of the following 6 protease inhibitors: leupeptin (0.5–4 μg/ml), l-trans-epoxysuccinyl leucylamido (4-guanidine) butane (1–8 μM), N-tosyl-l-phenylalanine chloromethyl ketone (0.1–0.4 mM), Nα-p-tosyl-l-lysine chloromethyl ketone (25–200 μg/ml), iodoacetic acid (0.5–4 mM), and phenylmethylsulfonyl fluoride (1–4 mM). These results suggest that a number of extrinsic and intrinsic factors may modulate excystation.