A procedure was developed for the rapid, analytical subcellular fractionation of entire homogenates from the Chinese hamster ovary and HeLa cell lines. The procedure avoids a nuclear sedimentation step and the losses that accompany such a step. A key to the development of this procedure was the addition to homogenates of either micrococcal nuclease or DNase I. Nuclease-treated homogenates were fractionated on self-forming Percoll gradients. The entire procedure from cell harvesting through collecting gradient fractions took only 2.5 h. The position of marker enzymes in the gradient fractions indicated clear resolution of plasma membranes, Golgi apparatus, endoplasmic reticulum, and lysosomes. This procedure should facilitate many studies requiring subcellular fractionation of cultured cells.