We have determined the influence of reference particle deformability and suspending buffer tonicity on the measurement of lymphocyte volume by an electronic particle volume analyzer. When the volume analyzer was standardized with latex spherules having a shape factor (fe) of 1.5, red cell volume was 96 cu micron and lymphocyte volume was 289 cu micron. The red cell volume corresponded closely to the true red cell volume; the true lymphocyte volume, however, was 218 cu micron when measured by the lymphocytocrit/lymphocyte count and 203 cu micron by wet lymphocyte weight and density (mean approximately 210 cu micron). The difference between the electronic volume (Ve) of 289 cu micron and true lymphocyte volume of 210 cu micron was due to the influence of lymphocyte deformability (shape factor) as it traverses the sizing aperture. Since the true volume equals the Ve/fe, the red cells with a shape factor near 1.0 were sized appropriately by this method. In contrast, the lymphocyte shape factor was 1.38; thus, the true lymphocyte volume was 289 cu micron/1.38 or 210 cu micron. The tonicity of the suspending solution also influenced the measurement of particle volume when osmotically inactive standard particles (e.g., latex spherules) were used as a reference. Whereas the true lymphocyte volume was 210 cu micron at 286 mosmole/liter, it was 194 cu micron at 330 and 229 cu micron at 250 mosmole/liter. The standard counting solution, Isoton, is hyperosmolar (330 mosmole/liter) and causes an 8% shrinkage of osmotically active cells.