A separation of spinach chloroplasts in vitro into fractions according to size (volume) and activity (light-dependent shrinkage and NADP reduction) has been achieved by stable-flow free boundary sedimentation-electrophoresis. The salient features of this chloroplast study are: (a) separation is achieved within 30 min; (b) only small density gradients are required, thus minimizing osmotic effects; (c) the fractions are collected continuously, with size fractionation being evidenced; and (d) particles are separated into fractions of higher and lower activities as compared with the control population.