Previous reports have claimed Adriamycin to be cytotoxic to cultured tumor cells when the drug is covalently immobilized on a solid support, thus suggesting a cell surface mechanism of action for the drug. Although these previous reports attempted to rule out released drug or endocytosis of drug-support particles as alternative explanations for the observed cytotoxicity, a more thorough analysis is necessary to substantiate fully the cell surface idea. In the present work, the stability of the drug-support linkage was increased by use of cross-linked polyvinyl alcohol as the support and cyanuric chloride or a diazonium salt for attachment of the drug. Different anthracycline orientations were tested by coupling Adriamycin at the amino sugar and carminomycin at the D-ring. The Adriamycin cross-linked polyvinyl alcohol and carminomycin cross-linked polyvinyl alcohol preparations had much lower drug release rates than did the earlier used carbamate-linked Adriamycin cross-linked agarose materials. All three immobilized drug preparations inhibited the growth of L1210 or S180 clones following 2- or 20-h incubation with cells at 37 degrees C. The results strongly support the concept that immobilized anthracyclines can be cytotoxic to cultured cells, for at least two different orientations of the drug on the support.