Electrocapture is a multifunctional microfluidic tool that can be used for concentration, sample cleanup, multistep reactions, and separation of biomolecules. Herein, we investigate the mechanisms underlying the electrocapture principle. A microfluidic electrocapture device was found to be capable of generating regions of different electric field, which are maintained in the flow by electric and hydrodynamic forces, with the zones of lower electric field strength upstream of those with higher strength. In addition to detection of the local electric fields by direct measurements, the existence of the zones was observed by the capture of a solution containing Coomassie and myoglobin. The two molecules were captured at different spots in a steady-state manner and were released (separated) at different electric fields. Considering these observations and the experimental values for the electric field strengths, flow velocities, and electrophoretic mobilities of DNA, proteins, and peptides, it is concluded that the macromolecules are captured between the field zones by a stacking mechanism.