The kinetics of hemolysis of erythrocytes in glycerol-containing media was studied spectrophotometrically. The hemolytic process starts by a rapid process, obeying a first order rate law, which is followed by a slow change in absorbance. The kinetics of hemolysis may be described by (a) the maximum absorption, Emax, due to cellular expansion, (b) the rate constant, k, of the fast process and (c) the final absorption at its end, Einf and the ratio Einf/Emax. At pH 6.85 in normal human cells, k = 0.72 min-1 while in hereditary spherocytosis cells, k = 1.06 min-1, iron deficiency k = 0.52 and beta-thalassemia minor k = 0.36 min-1. The percentages of Einf/Emax were 35.3 in control cells, while they were 9.8, 50.0 and 88.3 in spherocytosis, iron deficiency and thalassemia, respectively. Thus these kinetic parameters may help to distinguish and understand the above mentioned erythrocyte disorders. At physiological pH (7.4-7.2), no hemolysis was detected in the medium used. When the pH decreased, hemolysis occurred, its rate increasing gradually until pH 6.3. On further acidosis, the hemolytic rate slowed down again. Addition of DIDS to the whole blood prior to the test inhibits hemolysis. Similar effect of DIDS was noted in washed cells; this effect was partially reversed by albumin. These results suggest that a process involving band 3 affects the rate and degree of glycerol-induced hemolysis of normal red blood cells.