Cells in solid tumors generate an extracellular acidosis due to the Warburg effect and tissue hypoxia. Acidosis can affect the functional behavior of tumor cells, causing, e.g., multidrug resistance. In this process ERK1/2 and p38 mitogen-activated protein kinases (MAPK) seem to play a key role. However, the underlying mechanism of MAPK activation by extracellular acidosis remains unclear. Experiments were performed in three tumor and three normal tissue cell lines in which the cells were exposed to an extracellular pH of 6.6 for 3 h. Intracellular pH (pHi), protein expression and activation, acidosis-induced transactivation, and reactive oxygen species (ROS) formation were measured. Extracellular acidosis resulted in a rapid and sustained decrease of pHi leading to a reversal of the extra-/intracellular pH gradient. Extracellular acidosis led to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in three of six cell lines. Furthermore, p38 phosphorylation was also observed during sole intracellular lactacidosis at normal pHe. Acidosis-enhanced formation of ROS, probably originating from mitochondria, seems to trigger MAPK phosphorylation. Finally, acidosis increased phosphorylation of the transcription factor CREB and resulted in increased transcriptional activity. Thus, an acidic tumor microenvironment can induce a longer-lasting p38 CREB-mediated change in the transcriptional program.