A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.