In the present study, we investigated the role of actin filaments in the blastocoel (BC) of bovine blastocysts produced in vitro by using cytochalasin-D (CD), an inhibitor of actin polymerization. Blastocysts classified as good or poor based on their morphological normality were exposed to 2 μM CD for 1.5-2 hr. After incubation, the presence or absence of BC was observed, and the re-expansion rate was assessed after transferring CD-treated blastocysts with collapsed BC to a non-CD medium. Ultrastructural observation was also undertaken using a transmission electron microscope (TEM). The remaining blastocysts were immersed in a hypotonic solution of sodium citrate so that the cells could be counted to confirm the classification grade of blastocysts in this study. The percent of maintained BC under the presence of CD in the good group was significantly lower (P<0.05) than that in the poor group (3.3% versus 27.7%, respectively). The average cell number of blastocysts in the good group was significantly more (P<0.05) than that in the poor group. In addition, when the blastocysts with shrunken BC in both groups were cultured, re-expansion rates in the good and poor groups were 83.3 and 75.0%, respectively, and no significant difference was observed between groups. Based on observation of the ultrastructure with TEM, the microvilli on the surface of some trophoblast cells of some blastocysts in the poor group in the presence of CD showed a translucent matrix, and their electron density was low compared with that of trophoblast cells of blastocysts in the good and non-treated (control) groups. However, the electron density of microvilli after removal of CD in the poor group increased to a level comparable to those of the good and control groups. These results suggest that polymerizing actin may be required to sustain the blastocoel and microvilli of blastocysts produced in vitro. However, in poor grade blastocysts, the polymerization ability of actin present in the filamentous form in the microvilli in some cells might be lower than that in good grade blastocysts.