The role of actin filaments and contractions in hatching was determined in mouse blastocysts whose actin filament bundling abilities had been suppressed by H-89, an inhibitor of protein kinase A. The hatching rate of blastocysts developed from morulae in a medium containing H-89 at a concentration of 4.0 microM was 17.2%, which was significantly lower than the 76.7% of the control blastocysts developed from morulae in a medium without H-89. The rates of blastocysts starting hatching and forming a slit in the zona pellucida were significantly lower in H-89-treated blastocysts (84.4 and 21.9%) than in control blastocysts (100.0 and 90.6%). The lengths of time needed for slit formation in the zona pellucida and for completion of hatching were significantly longer in the H-89-treated blastocysts (27.4 and 43.3 h) than in the control blastocysts (6.5 and 18.8 h). Over the course of 32 h after blastocoel formation, the number of strong contractions was similar in the H-89-treated and control blastocysts, but the number of weak contractions was significantly fewer in the H-89-treated blastocysts (2.41 times) than in the control blastocysts (4.19 times). Although the distribution of actin filaments was similar in the H-89-treated and control blastocysts in the pre-hatching, hatching and post-hatching periods, the rate of H-89-treated blastocysts in which most trophectoderm cells possessed the fluorescence of actin filaments (12.7%) was significantly lower than the 95.0% of the control blastocysts in the pre-hatching period. These results suggest that actin filament-mediated movements of trophectoderm cells contribute to hatching by facilitating the protrusion of trophectoderm cells from a small hole in the zona pellucida and by enlarging the protrusion. We also suggest that the low hatching ability of the treated blastocysts is related to weak contractions with a low frequency and to strong contractions requiring a longer time for re-expansion.