A new class of radioiodinated biotin conjugated is described in which the amido bond between biotin and the labeled prosthetic group is reversed. One conjugate, (3-[125I]iodobenzoyl)norbiotinamide (4c, [125I]IBB) was labeled with Na125I in one step from (3-(tributylstannyl)benzoyl)norbiotinamide (4b, TBB) via a demetalation reaction. However, the analogous reaction with ((5-(tributylstannyl)-3-pyridinyl)carbonyl)norbiotinamide (6b, TPB) failed to yield ((5-[131I]iodo-3-pyridinyl)carbonyl)norbiotinamide (6c, [131I]IPB, necessitating a two-step approach for synthesizing [131I]IPB. The binding of [125I]IBB and [131I]IPB to streptavidin in vitro was identical to that of biotinyl-3-[125I]iodoanilide, a conjugate with an amido bond with normal configuration. Both [125I]IBB and [131I]IPB were stable in serum while the first-generation compound was rapidly degraded. The biodistribution patterns of [125I]IBB and [131I]IPB in mice are consistent with limited degradation of these conjugates by biotinidase and deiodinases.