The sedimentation coefficient of Cln varied inversely with the ionic strength in sucrose gradient ultracentrifugation. Natural antibody contamination was removed from impure Cln preparations by zonal ultracentrifugation in a high ionic strength (µ = 0.70) sucrose gradient, in which the Cln was 13.9S. The purified Cln gave a single precipitin line in Ouchterlony and immunoelectrophoresis with anti-whole nurse shark serum. Rabbit antibody against this preparation agglutinated cell-bound Cln, gave a single precipitin line of identity with purified Cln and whole nurse shark serum, and single precipitin arcs immunoelectrophoretically. At 0°C, 0.04 M EDTA inactivated Cln slowly and did not prevent Cln-site formation. EDTA inactivation was irreversible. In the presence of EDTA, immunoelectrophoresis of purified Cln gave three precipitin arcs. Esterase activity, present in the partially purified (urea-precipitated) Cln preparations, was absent in purified Cln with N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester, or N-carbobenzoxy-l-tyrosine p-nitrophenyl ester as substrates.