Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel. We tested whether a gradient of intracellular Ca2+-concentration ([Ca2+]i) underlies the horizontal polarization of K+ channel activity. [Ca2+]i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca2+]i indicated that a horizontal gradient exists, with [Ca2+]i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca2+]i in the cell body were found to be 135 +/- 34 and 405 +/- 59 nml/l, respectively, whereas they were 79 +/- 18 and 307 +/- 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K+ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca2+]i to test whether its maintenance is due to an uneven distribution of Ca2+ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La3+ alone or together with charybdotoxin, a specific blocker of Ca2+-sensitive K+ channels, slowed migration to 47 +/- 10% and 9 +/- 5% of control, respectively. Local blockade of Ca2+ influx into the cell body and the lamellipodium with la3+ was followed by a decrease of [Ca2+]i at both cell poles. This points to Ca2+ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn2+ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca2+]i. We hypothesize that the polarized distribution of intracellular Ca2+ stores contributes significantly to the generation of a gradient of [Ca2+]i.