The volumes of the major tissue compartments, vascular space, interstitial space, and luteal cells, in the corpus luteum (CL) of 16‐day pregnant rats were determined morphometrically after different fixation procedures. Ovaries were fixed by perfusion at physiologic pressures (PF), by immersion directly in fixative (IF), by immersion after clamping blood vessels to prevent escape of blood (IFC), or by immersion after administration of heparin to prevent blood clotting (IFH). Three CL in each of eight ovaries were examined for each fixation method. Total volumes were significantly larger in CL fixed by PF and IFC, and approximated physiologic volumes as assessed from frozen ovaries. Perfusion‐fixed CL had significantly larger vascular and interstitial spaces and smaller cell volumes than immersion‐fixed CL. Capillary surface area varied significantly between the four groups in accordance with absolute volume of vascular space. Estimated numbers of endothelial cells and amounts of connective tissue differed significantly, presumably due to difficulties in identifying these components in immersion‐fixed CL with collapsed vessels. The fixation methods used caused substantial fluid shifts between the three major compartments. A theoretical approach for considering these shifts and the likely physiologic situation was developed.