Klaus Hofmann,* Gail Titus, Judith A. Montibeller, and Frances M. Finn abstract: The use of biotinylated hormones as ligands on avidin-Sepharose columns represents an attractive approach to the isolation of receptors. However, the strong noncovalent interaction between the hormone and its receptor on one hand and biotin and avidin on the other poses major obstacles to receptor retrieval. We report the results of a systematic search for weaker binding biotin containing ligands that are displaceable by biotin. Syntheses are described of the N-hydroxysuccinimido esters of iminobiotin, dethiobiotin, 6-(iminobiotinylamido) hexanoic acid, and 6-(dethiobiotinyl-amidojhexanoic acid. These active esters served to acylate A “’A ‘, Af'B29-Boc2-insulin (from porcine insulin), and the re-sulting protected insulins were converted to A “, B ‘-imino-biotinyl-, A “’B'-dethiobiotinyl-, A “’B'-[6-(iminobiotinyl-amido) hexyl]-, and A “’B'-[6-(dethiobiotinylamido) hexyl]-insulins, respectively. Complexes of these insulin derivatives with avidin and succinoylavidin were prepared, and the rate of their dissociation (off-reaction) was determined in the presence of [14C] biotin. It was found that the attachment of the insulin molecule to biotin and the biotin analogues exerted a striking effect on the rate of dissociation of the complexes. The half-time for dissociation of the avidin-biotin complex is 200 daysand that of the succinoylavidin-biotin complex is 127 days. The complex of biotinylinsulin with avidin or