The frequency of occurrence of vacuoles in red blood cells was studied by transmission electron microscopy. Small vacuoles were found in about 13% of the cell sections, and they had a mean diameter of 130 +/- 72 nm (mean +/- SD). It can be estimated that there were about 20 small vacuoles per erythrocyte. The frequency of vacuoles was similar in density-separated cell fractions. In splenectomized patients, the small vacuoles were 4 times more frequent; there was again no difference in vacuole density between top and bottom fractions of density-separated red blood cells. The bottom fraction of red blood cells from splenectomized patients, however, had a high incidence of large vacuoles (greater than 300 nm in diameter) and clustering of small vacuoles. These large vacuoles were probably the result of aggregation and fusion of small vacuoles, and their size allowed detection by light microscopy. Hence, the well-known "pocked" or "pitted" red blood cells of splenectomized individuals were more frequent in the bottom fraction. We conclude that small vacuoles occur normally in erythrocytes, that they tend to cluster and fuse during cell aging, and that the spleen is capable of removing these structures when they reach a certain size.