Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedione), a peptide analogue originally isolated from human urine and serum, appears to have antineoplastic activity. In view of the close resemblance of the structure of A10 to that of DNA intercalative anticancer drugs, spectroscopic studies were performed to determine whether its mode of action could similarly involve binding to DNA. DNA thermal denaturation studies demonstrated that A10 was capable of interacting with DNA in a specific manner; of the synthetic polynucleotides employed in this study, A10 had the greatest effect on poly(dA-dG).poly(dC-dT), suggesting some sequence preference. However, ultraviolet and fluorescence spectroscopic studies demonstrated that interactions of A10 with DNA were weak in comparison to those of classical intercalating agents. Mass spectroscopic studies suggested that A10 did not react covalently with DNA. The weak yet apparently specific interaction of A10 with DNA indicates that the mode of action of A10 may involve binding to chromatin, facilitated by nuclear protein receptors analogous to steroid and thyroid hormones.