Antibodies against biotinylated proteins have been identified in 10% of individuals tested (6/60). These antibodies bind readily to biotinylated proteins (50% inhibitory concentration = 0.59 mumol/L) but only modestly to free biotin or biocytin. It is unlikely that any clinical consequences occur as a result of these antibodies, because the affinity for free biotin is too low (50% inhibitory concentration = 0.51 mmol/L) to affect the normal level of free plasma biotin, 0.5 nmol/L. The pathogenesis of this antibiotin immune response is unclear. Repeat testing of several individuals 5 months apart indicated that the antibiotin response was stable. In addition, 51 of the individuals tested for antibiotin antibodies were also examined for antiavidin antibodies. Whereas five were positive for antiavidin, only one individual was positive for both antibiotin and antiavidin antibodies. The presence of an antibiotin antibody is unlikely to affect the in vivo use of biotinylated proteins or cells in human subjects because its affinity for biotinylated proteins is modest and the level of biotinylation for in vivo studies is intentionally low. However, these antibodies may affect clinical or laboratory assays based on the biotin-avidin system where an antibiotin antibody may either positively or negatively affect the specific assay.