As a consequence of poor perfusion and elevated acid production, the extracellular pH (pHex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pHex of 7.4. This is significant, because slight changes in pHex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive potential of two human melanoma cell lines; the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHin) and intracellular calcium ([Ca2+]in), we examined the effect of these parameters on invasion. While changes in [Ca2+]in were not consistent with invasive potential, the changes in pHin were. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.