Infections with parvovirus B19 may lead to apoptosis of affected cells with anemia, acute fulminant liver failure, and placental insufficiency. The apoptosis is presumably caused by the viral protein NS1 which is known to trigger a signaling cascade eventually leading to activation of caspases. In several cell types apoptosis is paralleled by cytosolic acidification, which may be secondary to inhibition of the Na+/H+ exchanger and may support activation of pH sensitive caspases and endonucleases. However, nothing is known about effects of NS1 on Na+/H+ exchanger activity and cytosolic pH. The present study thus explored the effect of NS1 expression on cytosolic pH (pHi) and Na+ dependent realkalinization ([Delta]pHi) following an ammonium pulse. Overexpression of NS1 in human retinoblastoma cells (RXR-SW) led to a significant decline of pHi and to a significant decrease of [Delta]pHi without affecting appreciably cellular buffer capacity. The decrease of pHi and of [Delta]pHi following NS1 expression could be significantly blunted by inhibition of tyrosine kinases with herbimycin (10 mM), lavendustin (10 mM), and genistein (10 mM), and inhibition of caspase 3 with zVAD (10 mM). In vitro experiments revealed that caspase 3 degrades NHE1. In conclusion, NS1, probably via tyrosine kinases, activates caspase 3, which in turn degrades NHE1.