The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (> 95% pure), exhibiting an Arg residue as their C-terminal (site P-1) for trypsin biorecognition, a Pro or Ala in site P-2, and a Thr or Val in site P-3. Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 mu mol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 mu mol/g gel. The K-D values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K-D values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K-D 8.7 mu M), whereas the sequence H-TAR-OH displayed the lowest (K-D 38 mu M). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 mu mol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield.