The aim of this study was to investigate the hypothesis that saturated fatty acids are differentially cytotoxic to cancer cells. Three studies were undertaken to: (1) measure the toxicities of stearic and oleic acids to normal and malignant cells in vitro, (2) assess if there is any relationship between toxicity and relative fatty acid composition and (3) determine whether the relative fatty acid composition of a cancer cell line could be modified by sterculic acid, an inhibitor of delta-9-desaturase. Stearic (18:0) and oleic (18:1) acids inhibited the colony-forming abilities of five human cancer cell lines and two non-neoplastic cell lines in a dose-dependent fashion. The concentration of oleic acid required to reduce colony formation ability by 50% was 2.5–6.0-fold greater than that of stearic acid. Addition of sterculic acid to a cancer cell line resulted in steady-state levels of stearic acid and increasing percentage of oleic acid.