Cultured, human endothelial cells from the iliac vein were divided into two groups for rapid-freezing using liquid helium in a Heuser-type cryopress freezing machine. The first group of cells was prefixed in buffered 2% glutaraldehyde solution whereas the second group was freshly-frozen. Following freeze substitution and embedding, morphometric analysis was carried out on thin sections of groups of endothelial cells. Measurements showed that glutaraldehyde prefixation significantly increases the numerical and volume densities of the vesicles compared to those of the freshly-frozen cells. However, the fact that no significant increase was demonstrated either in the numerical or volume densities of the caveolae, and that no significant change was observed in the ratio of the cell perimeter to area, suggests that the membrane source for these extra vesicles is internally generated. Studies are currently under way to identify the source of this membrane.