Dimethyl-DL-2,3-distearoyloxy-propyl-2'-hydroxy-ethylammonium++ + (Rosenthal's inhibitor) was coupled to carboxyhexyl-Sepharose 4B, through carbodiimide chemistry. Phospholipase A2 from Heloderma horridum horridum and Crotalus adamanteus bind to the immobilized ligand in the presence of Ca2+ and can be easily eluted under acidic conditions or in the presence of a chelating agent, respectively. This affinity media proved to be effective also in the purification of a Ca2(+)-independent phospholipase A1 from vespid venom.