A technique based on the measurement of streaming potentials has been developed to evaluate the electrokinetic region of the cell periphery. This approach is feasible for cell lines propagated in in vitro cell culture in monolayer form. The advantage of this system is that cells may be evaluated in the living state attached to a substrate. Thus it is not necessary to subject the cells to enzymatic, chemical, or mechanical trauma required to obtain monodisperse suspensions which are then normally evaluated by microelectrophoresis. In this manner it should be possible to study the influence of substrate and environmental factors on the charge density and potential at the cell periphery. The apparatus and procedure are described as well as some results concerning the electrokinetic (ζ) potential of borosilicate capillaries as a function of ionic strength, pH, and temperature. The effect that turbulence and entrance flow conditions have on accurate streaming potential measurements is discussed. The ζ‐potential of BALB/c 3T12 fibroblasts has been quantitated as a function of pH, ionic strength, glutaraldehyde fixation, and Giemsa staining.