Dark-grown corn coleoptiles and parsley hypocotyls and their roots were loaded with acetoxymethyl esterified forms of the Ca2+ indicator fluo-3, and the pH indicator 2',7'-bis (2-carboxyethyl)-5(and-6)-carboxyfluorescein. These tissues were treated with the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin analogue, or abscisic acid (ABA), and the cytosolic pH (pHcyt) and cytosolic Ca2+ ([Ca2+]cyt) changes were monitored by confocal scanning optical microscopy. Over a period of 4 min pHcyt decreased 0.1-0.2 pH unit and [Ca2+]cyt increased from 280 to 380 nM in response to 2,4-D. ABS, on the other hand, induced cytosolic alkalinization of 0.05-0.1 pH unit with a concomitant increase in [Ca2+]cyt from 240 to 320 nM over a 4-min period. Responses similar to these were observed in all the tissues tested. We suggest that pHcyt profoundly influences signaling by[Ca2+]cyt, possibly by regulating Ca2+-protein binding, and that the divergent effects of auxin and ABA on pHcyt underlie their mutual antagonism.