Although it has long been appreciated that larger eukaryotic cells have larger nuclei, little is known about how this size relationship is maintained. Here we describe a method for measuring the aqueous volume ratio of nucleus to cytoplasm, two compartments which are interconnected via the pores in the nuclear envelope. We then use that method to identify proportional cellular dimensions in variously treated cells and in different cell types. Cells were scrape loaded with a mixture of fluorescent dextrans: Texas red dextran, average mol wt = 10,000 (TRDx10), and fluorescein isothiocyanate dextran, average mol wt = 70,000 (FDx70). After introduction into the cytoplasmic space, the TRDx10 distributed into both the nucleus and cytoplasm, whereas the FDx70 was restricted to cytoplasm, due to size exclusion by the nuclear pores. The aqueous nucleocytoplasmic volume ratio (RN/C) was determined by measuring, from fluorescence images of spread cells, total cellular fluorescence of each of the two probes and the fluorescence ratio of those probes in the cytoplasm. RN/C was unaffected by the measurement procedure or by varying temperatures between 23 degrees and 37 degrees C. Loading excess unlabeled dextrans had little effect on RN/C, with the single exception that high concentrations of large dextrans could lower RN/C in endothelial cells. Expanding intracellular membranous compartments of macrophages by phagocytosis of latex beads decreased RN/C. Expanding the same compartment by pinocytosis of sucrose, which nearly doubled total cell volume, had little effect on RN/C, indicating that nuclear volume was more closely linked to the cytoplasmic volume, exclusive of vesicular organelles, than to total cell volume. RN/C was the same in mononucleate and binucleate endothelial cells. Finally, measurements of RN/C in murine bone marrow-derived macrophages, bovine aortic endothelial cells, Swiss 3T3 fibroblasts, PtK2 cells, and CV-1 cells revealed that nuclear volume scaled allometrically with cell volume. The allometric relationship indicated that cell volume was proportional to nuclear surface area.