Here we describe the use of pulse-mode scanning ion conductance microscopy (SICM) to observe volume changes and cell membrane movements during the locomotion of cultured cells in the range of minutes to several hours. The microscope is based on the pulse-mode SICM previously developed for stable imaging of single cells in culture. Our instrument uses current pulses to control the distance between cell surface and electrode tip as well as a back-step mode to prevent contact of tip and membrane during lateral movements of the probe. We performed repeated scans of cell surfaces using feedback-controlled piezoactors to position the electrode. Using patch-clamp-type electrode tips the height of cells could reproducibly be measured with a standard deviation of 50 nm. To quantify and separate changes in cell position and volume occurring between consecutive scans, a program was written to subtract images and calculate volume changes. Examples of repeated scans show that membrane movements in the range of 30 min to a few hours can be quantitatively monitored with a lateral resolution of 500 nm using difference images and that faster movements in the range of minutes can be recorded at defined cell sections using the line scan mode. Difference images indicate that volume changes can affect cell surfaces inhomogeneously, emphasizing the role of the cytoskeleton in the stabilization of cell shape.