Arachidonic acid (AA) is often utilized in in vitro studies to label cellular pools of AA or to elicit cellular responses dependent on eicosanoid production. Because of the hydrophobic nature of AA, organic diluents such as ethanol or dimethyl sulfoxide are utilized in preparing concentrated solutions. The fate of AA when added to aqueous medium is not generally considered because of the dilution of the AA, although some investigators utilize bovine serum albumin (BSA) to solubilize as well as to trap AA and its hydrophobic metabolites. These experiments demonstrate a rapid and progressive decline in AA concentration when added to aqueous media in tissue baths and in glass test tubes. The extent of the decline was greater in the tissue baths than in the test tubes. The calcium ionophore A23187, which is used to stimulate AA metabolism, is also hydrophobic, and its concentration also decreased when added to aqueous media. The decline in the concentration of both AA and A23187 was due to adsorption to the container walls. The presence of 1% BSA in the aqueous solution attenuated and even eliminated the decline in the concentration, indicating binding of the two agents to the protein. However, the presence of BSA in culture medium inhibited the A23187-induced stimulation of AA metabolites in baboon aortic smooth muscle cells. These results underscore the complexities arising from the in vitro use of hydrophobic substances in biological systems.