Two-cell bovine embryos become arrested in development when exposed to a physiologically relevant heat shock. One of the major ultrastructural modifications caused by heat shock is translocation of organelles toward the center of the blastomere. The objective of the present study was to determine if heat- shock-induced movement of organelles is a result of cytoskeletal rearrangement. Two-cell bovine embryos were cultured at 38.5°C (homeothermic temperature of the cow), 41.0°C (physiologically relevant heat shock), or 43.0°C (severe heat shock) for 6 h in the presence of either vehicle, latrunculin B (a microfilament depolymerizer), rhizoxin (a microtubule depolymerizer), or paclitaxel (a microtubule stabilizer). Heat shock caused a rearrangement of actin-containing filaments as detected by staining with phalloidin. Moreover, latrunculin B reduced the heat-shock-induced movement of organelles at 41.0°C but not at 43.0°C. In contrast, movement of organelles caused by heat shock was inhibited by rhizoxin at both temperatures. Furthermore, rhizoxin, but not latrunculin B, reduced the swelling of mitochondria caused by heat shock. Paclitaxel, while causing major changes in ultrastructure, did not prevent the movement of organelles or mitochondrial swelling. It is concluded that heat shock disrupts microtubule and microfilaments in the two-cell bovine embryo and that these changes are responsible for movement of organelles away from the periphery. In addition, intact microtubules are a requirement for heat-shock-induced swelling of mitochondria. Differences in response to rhizoxin and paclitaxel are interpreted to mean that deformation of microtubules can occur through a mechanism independent of microtubule depolymerization.