To understand developmental mechanisms, it is important to know when and where signaling pathways are activated. The spatio-temporal pattern of activation of mitogen-activated protein kinase (MAPK/ERK) was investigated during embryogenesis of the ascidian Halocynthia roretzi, using an antibody specific to the activated form of MAPK. During cleavage stages, activated MAPK was transiently observed in nuclei of the precursor blastomeres of endoderm, notochord, mesenchyme, brain, secondary muscle, trunk lateral cells and trunk ventral cells. These sites of MAPK activation are consistent with results of previous studies that have analyzed the embryonic induction of various tissues, and with results of inhibition of MAPK kinase (MEK) in ascidians. Activation of MAPK in notochord and mesenchyme blastomeres was observed in a short period in a single cell cycle. In contrast, in brain and secondary muscle lineages, MAPK activation spanned two or three cell cycles, and upon each cleavage, MAPK was asymmetrically activated in only one of the two daughter cells that remained brain or secondary muscle lineages. During later stages, MAPK activation was predominantly observed in the central nervous system. A conspicuous feature at this stage was that activation appeared to alternate between positive and negative along the anterior-posterior axis of the neural tube. During the tail elongation stage, MAPK was quiescent.