The antitumor drug Adriamycin, when preincubated with human red blood cells (discocytes) for 10 min, prevented the formation of echinocytes induced by the calcium ionophore A23187 in the presence of 0.2 mM calcium. The degree of protection was concentration dependent and was greater than 90% at 10 microM Adriamycin. Adriamycin did not interfere with the accumulation of calcium induced by a 5 microM concentration of the ionophore. Adriamycin reversed echinocyte morphology to the discocyte form in echinocytes which had been formed by adenosine triphosphate depletion but not those formed after treatment with A23187 and Ca2+. Its ability to protect against Ca2+-induced echinocyte formation contrasts with the failure of the local anesthetic procaine to exert such an effect, even at 45 mM (J. Palek et al., Blood, 50: 155-164, 1977), and this difference suggests that Adriamycin may not be acting simply as a chaotropic agent. This hypothesis was supported by the observation that Adriamycin alone did not induce a cup-form morphology in discocytes (stomatocytosis). Wheat germ agglutinin protection of echinocyte formation induced by calcium loading was reversed by 30 mM N-acetylglucosamine, which partially reversed the Adriamycin protection of echinocyte formation. However, desialylation of human red blood cells with Clostridium perfringens type V neuraminidase, while preventing the protection of echinocyte formation by wheat germ agglutinin, had no effect on the protection afforded by Adriamycin. This suggests that Adriamycin does not prevent echinocyte formation via binding to the sialic acid residues of the transmembrane protein glycophorin and that another mechanism or mechanisms are involved in its action to modulate morphological transitions of the red blood cell membrane.