Polyethylene glycol-conjugated adenosine deaminase (pegademase) is used for enzyme replacement therapy for patients with severe combined immunodeficiency caused by adenosine deaminase deficiency. The entrapment of pegademase within human energy-replete carrier erythrocytes using a hypo-osmotic dialysis procedure was investigated with the objective of prolonging the in vivo circulatory half-life of the enzyme and maintaining therapeutic blood levels. Native unmodified adenosine deaminase (ADA) was similarly studied. The efficiency of pegademase entrapment was low (9%) whereas the entrapment of native unmodified ADA was substantial (50%), suggesting that the polyethylene glycol side-chains were impeding intracellular entrapment. The biochemical characteristics and the osmotic fragility of these carrier erythrocytes were not adversely affected by the entrapment of either pegademase or native ADA. In vivo survival studies of pegademase-loaded 51Cr-labelled carrier erythrocytes in an ADA-deficient adult patient showed a mean cell half-life of 16 d. Carrier erythrocyte-entrapped pegademase and native ADA had in vivo half-lives of 20 and 12.5 d, respectively, demonstrating that entrapment prolongs the half-life over that of plasma pegademase, which has a circulating half-life of 3-6 d. These results provide the basis for a more extensive clinical evaluation of carrier erythrocyte-entrapped native adenosine deaminase therapy.