A new, sensitive radioassay for the determination of biotinidase activity was developed which measures the release of [14C-carboxyl]-p-aminobenzoate from N-biotinyl-[14C-carboxyl]-p-aminobenzoate. Biotinidase activity in serum from normal individuals is comparable to that determined by the colorimetric assay, but the radioassay is approximately 100 times more sensitive. Biotinidase deficiency was confirmed in the serum of patients who were previously shown to have reduced activities by the colorimetric assay. Although biotinidase activity was not detectable in extracts of normal peripheral blood leukocytes and fibroblasts using the colorimetric assay, activities could be measured by the radioassay. Using this method we demonstrated deficient biotinidase activity in extracts of leukocytes and fibroblasts from affected patients.