High-density lipoprotein (HDL) cholesterol levels have an inverse relationship with the frequency of coronary and cerebrovascular disease. Most commonly HDL deficiency is environmentally modulated. Familial hypoalphalipoproteinemia (FHA) is a genetically determined HDL deficiency disease, in all likelihood transmitted as an autosomal dominant trait and associated with premature atherosclerosis. Apolipoprotein AI (apo AI) is the major apoprotein in the HDL particle, and defects in this protein have been suggested as the cause of FHA. We have identified a large family of Spanish descent with FHA and performed genetic linkage analysis using restriction fragment length polymorphisms in the Apo AI-CIII-AIV gene cluster to test this hypothesis. Results in this family formally exclude the apo AI-CIII-AIV gene cluster as the site for the mutation underlying FHA.