The presumptive muscle cells (B5.1 blastomeres) were isolated from 16-cell-stage embryos of the ascidian, Ciona intestinalis. The isolated cells were allowed to divide either twice or three times thereafter. Then further divisions of the cells were continuously inhibited by a simultaneous treatment with aphidicolin (a specific inhibitor of DNA synthesis) and cytochalasin B (an inhibitor of cytokinesis). When development of muscle-specific acetylcholinesterase in these division-arrested progeny cells of B5.1 blastomeres was examined histochemically, the B5.1 blastomeres which had been allowed two further divisions did not produce any detectable acetylcholinesterase activity. Whereas those which had been allowed three further divisions showed the tissue-specific enzyme activity. These results provide further evidence for the presence of a quantal DNA replication cycle for the tissue-specific enzyme development, which is qualitatively different from the other DNA replication cycles.