The retention and elution of acetylcholinesterase from bovine erythrocytes and electric eel on N-methylacridinium affinity columns have been compared at various ligand concentrations. A soluble 7.7 S dimeric form of bovine erythrocyte acetylcholinesterase required a ligand concentration of 2.0-2.8 mumol/ml in 0.1 M NaCl for retention, compared to 0.44 mumol/ml for various forms of the electric eel acetylcholinesterase. The difference in the retention of acetylcholinesterase from these two sources could not be explained by differences in their oligomeric structure. The affinity of bovine erythrocyte acetylcholinesterase for N-methylacridinium was 13-fold or more lower than the electric eel acetylcholinesterase at similar ionic strengths. N-Methylacridinium appeared to react selectively with the catalytic anionic site of both enzymes. It was concluded that the affinity of the side arm ligand was the major determinant of the differences in the retention properties of the eel and erythrocyte acetylcholinesterase. The difference in affinity for N-methylacridinium probably reflects differences in the organic cation binding region of the two enzymes.