Dynamic field gradient focusing uses an electric field gradient to separate and concentrate proteins in native buffers. A prototype preparative-scale dynamic field gradient focusing apparatus reproducibly separated hemoglobin and bovine serum albumin with a mean resolution of 2.64 +/- 0.503. Run-to-run variations in the hemoglobin's focal point and peak width appeared to be related to fluctuations in the shape of the electric field, rather than the 5% accuracy of the pump that provided the counter-flow in the separation annulus. The variation in the electric field gradient was probably due to the formation and expansion of an ion-depleted region at the top of the separation annulus.