The goal of this work was to find a method suitable for the extraction of adenine nucleotides from cultured vascular endothelial cells. Extraction of cell monolayers with 80% methanol in water yielded extracts with a higher content of ATP than did extraction of cells with perchloric acid, trichloroacetic acid, or boiling water. The optimal extraction solution was 80% methanol with 0.5 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or EDTA, heated to 70 degrees C immediately before use. Extraction of nucleotides by this solution was rapid and the recovery of exogenous ATP added during the extraction process was generally greater than 90%. An aqueous methanol or ethanol solution may be applicable for the extraction of nucleotides and other metabolites from cultured animal cells, dispersed cells, and frozen, powdered tissues.