Previous observations in rat two-cell embryos suggested that distribution of microfilaments and microtubules are involved in developmental arrest. Therefore, we examined the distribution of cytoskeletal proteins, actin binding proteins, and microtubule-associated proteins in rat two-cell embryos. We also examined gene expression of beta-actin, alpha-tubulin, and cytoskeletal proteins that showed changes in their distributions. Distribution of cytoskeletal proteins was examined by immunocytochemistry. Although distributions of alpha-actinin, MAP1A, MAP1B/MAP5, and MAP2 were disturbed in arrested embryos, these abnormal distributions occurred following the initiations of developmental arrest and marked damage of microfilaments and microtubules. Gene expression of cytoskeletal proteins was examined by RT-PCR. Beta-actin and alpha-actinin mRNA was detected in normal late two-cell stage but not in arrested embryos. The difference occurred after zygotic gene activation. Expression of alpha-tubulin was detected in neither normal late two-cell stage nor arrested embryos. No MAP1A, MAP1B/MAP5, or MAP2 expression was detected in embryos during the two-cell stage. In conclusion, both distributions of microfilaments and microtubules are closely involved in rat developmental arrest, but other distributions of cytoskeletal proteins, actin binding proteins, and microtubule-associated proteins do not appear to have major roles in two-cell arrest. Furthermore, mRNA expression patterns are different between microfilaments and microtubules. Both distribution and mRNA transcription of microfilaments are involved in rat developmental arrest, whereas only distribution of maternal microtubules is disturbed in arrested embryos.