Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific "puffs" in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5 x 10(-6) mol/1 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca(2+)-dependent increase of nuclear volume by 23.0 +/- 2.3%. Amiloride, a blocker of plasma membrane Na+/H+ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9 +/- 3.9%. Measurements of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23 +/- 0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amiloride-sensitive plasma membrane Na+/H+ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.