Electrophoretic and fingerprinting studies in a patient with congenital hemolytic anemia revealed the presence of the unstable hemoglobin Köln (β98val→met). Examination of parents and siblings gave normal results. Extensive blood group and isozyme studies were consistent with the thesis that Hb Köln disease in the propositus was the result of a fresh mutation in one of his parent’s gametes. In the propositus, the activities of enzymes of the Embden-Meyerhof and pentose phosphate pathways were increased, but the level of ATP was decreased. Methemoglobin reduction was delayed when the NADPH-dependent system was utilized with added methylene blue and gave a false-positive result in the glucose-6-phosphate dehydrogenase screening test. Methemoglobin reduction in the absence of methylene blue was normal. Increased methemoglobin and Heinz body formation, decreased osmotic fragility, decreased red cell deformability, and a disproportionate potassium loss without sodium gain occurred with metabolic depletion. The rate of decline of glutathione in propositus’ cells paralleled that in normal cells. Autologous survival of Hb Köln cells was decreased but was not compromised further by oxidant drugs. Marked splenic sequestration of Hb Köln erythrocytes was demonstrated, and an excellent response to splenectomy with improved erythrocyte survival was observed. The intracellular precipitation of unstable globin chains, intracellular dehydration, and increased membrane rigidity probably all contribute to the splenic entrapment of these erythrocytes.