Solid growing tumors often show pronounced extracellular acidosis which influences intracellular signal cascades and tumor phenotype, for example invasiveness. We analysed the impact of acidosis on invasiveness of AT1 cells which had been exposed either to a control (pH 7.4) or an acidic extracellular environment (pH 6.6) for 3–6 h prior to assessment. Invasiveness was monitored on the one hand by the ability of AT1 cells to disrupt a tight epithelial barrier (formed by MDCK-C7 cells), using transepithelial resistance (Rte) as measure. Furthermore, invasiveness was monitored by the ability of AT1 cells to cross a matrigel. AT1 cells led to a substantial reduction of Rte from the basolateral but not from the apical side, irrespective of the extracellular pH. This effect did not depend on matrix metalloproteases nor on cathepsine B (whose protein activity in the media was detected by gelatine zymography and fluorometric assay). In contrast, the ability to cross a matrigel is enhanced by extracellular acidosis, an effect abrogated by inhibition of ERK1/2- and p38 signaling. These findings indicate that acidosis stimulates invasiveness of AT1 cells at the level of migration into the surrounding matrix. This effect is mediated by p38 kinase and ERK1/2 signaling.