To detect high density lipoprotein binding proteins, a new ligand blotting method that uses apolipoprotein AI-dinitrophenol as a ligand was established. Apolipoprotein AI-dinitrophenol was prepared by incubating the mixture of apolipoprotein AI and 2,4-dinitrobenzene sulfonic acid sodium salt at pH 8.5. Four to six dinitrophenol groups were found to be conjugated w,ith an apolipoprotein Al molecule without any obvious impairment of its binding activity to the binding proteins. By this detection procedure, we detected two apolipoprotein Al binding proteins in plasma membrane fractions from bovine liver and adrenal cortex with molecular masses of 120 kDa and 95 kDa. Since this new ligand blotting method is simple and time-saving, it is expected to become a useful tool in the studies on high density lipoprotein binding proteins. (C) 1995 Academic Press, Inc.