Clathrin-associated proteins purified from bovine brain exhibited an ultraviolet spectrum with absorbance maximum at 256 nm and were found to contain tightly bound nucleotide. This nucleotide was identified as AMP and/or ADP by thin-layer and high-performance liquid chromatographic analyses. The phosphorylation state of the bound nucleotide varied with storage conditions, suggesting that exchange with ATP might occur while a molar ratio of two nucleotides per protein molecule is maintained. This nucleotide binding site may play a role in the functions of clathrin-associated proteins.