AbstractThe anchorage‐dependent and anchorage‐independent growth of the human mammary carcinoma cell line MDA‐MB‐231 is inhibited by vitamin A (retinol). Clones resistant to growth inhibition by retinol were isolated from this cell line in soft agar without the use of mutagens. This paper describes the isolation and characterization of the resistant lines. The clones were selectively resistant to retinol. There was significant growth inhibition after treatment with retinoic acid and 13‐cis‐retinoic acid. The resistant clones maintain their resistance to retinol through multiple passages. Resistance is specific for inhibition of growth, because treatment of the resistant clones results in stimulation of plasminogen activator activity without alteration of proliferation. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis shows no significant qualitative or quantitative difference in the clones when compared with the MDA‐MB‐231 parent line. Although the clones do not regrow in soft agar, they are tumorigenic in athymic mice. Tumors are produced at a rate similar to the parent line. The advantage of this isolation method is that sensitive and resistant malignant cells derived from the same parent cell line are now available to study the molecular events involved in the inhibition of cellular proliferation after treatment with retinol.