We investigated whether alterations in the mechanisms involved in intracellular pH (pH in ) and intracellular calcium ([Ca 2/ ] in ) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pH in and [Ca 2/ ] in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steadystate pH in and [Ca 2/ ] in between these cell types were not significantly different. Treatment of cells with NH 4 Cl resulted in larger pH in increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H / buffering capacity than A375P. NH 4 Cl treatment also increased [Ca 2/ ] in only in C8161 cells. To determine if the changes in [Ca 2/ ] in triggered by NH 4 Cl treatment were due to alterations in either H / -or Ca 2/ -buffering capacity, cells were treated with the Ca 2/ -ionophore 4Br-A23187, to alter [Ca 2/ ] in . The magnitude of the ionophore-induced [Ca 2/ ] in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced [Ca 2/ ] in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca 2/ ] in regulatory mechanisms than C8161 cells, to recover from Ca 2/ loads. Removal of extracellular Ca 2/ ([Ca 2/ ] ex ) decreased [Ca 2/ ] in in both cell types at the same extent. Ionophore treatment in the absence of [Ca 2/ ] ex transiently increased [Ca 2/ ] in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca 2/ -ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca 2/ ] in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca 2/ stores for [Ca 2/ ] in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H / -ATPases, corroborated our previous results that V-H / -ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells (MartıB nez-ZaguilaB n et al., 1993). Collectively, these data suggest that distinct pH in and [Ca 2/ ] in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.