A reproducible, high-yield high-performance gel-permeation chromatographic method for proteins was developed and applied to the final step of purification of human serum biotinidase. One diol-type silica gel column, Tosoh TSK-gel 3000 SW (300 mm x 7.9 mm I.D.; average pore size 30 nm), and two Jasco Bio-Fine GFC SI 150-K columns (300 mm x 7.9 mm I.D.; average pore size 15 nm) were connected in series. A 0.1 M sodium phosphate buffer (pH 6.0) solution containing 0.3 M sodium chloride, glycerol (2.5%, v/v) and the non-ionic detergent Nonidet P-40 (NP-40, 0.15%, v/v) was utilized as an eluent. Recovery of the protein bovine serum albumin from the separating columns was measured by a spectrophotometric method and found to be 77.0 +/- 3.74% (mean +/- S.D.). The recovery of the total activity of human serum biotinidase was 72.6 +/- 13.0%. Since biotinidase activity was not recovered from the column in the absence of NP-40, the introduction of this non-ionic detergent to the mobile phase was shown to be essential for the final purification step of human serum biotinidase.