Incubation of Chinese hamster ovary cells (CHO-K1) with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol (0.1 microM) in lipid-deficient medium led to a major change in cellular sterol composition, which was characterized by a very marked accumulation of C30 sterols (lanosterol and 24,25-dihydrolanosterol). The accumulation of C30 sterols was associated with a striking change in cell morphology. The change in cell shape (elongation) was similar to that described previously (A. W. Hsie and T. T. Puck, 1971. Proc. Natl. Acad. Sci. USA. 68: 358-361; and confirmed herein) for CHO-K1 cells incubated in the presence of dibutyryl cAMP (1 mM). This change in morphology, induced by dibutyryl cAMP, was not accompanied by a change in cellular sterol composition. The cell elongation and accumulation of C30 sterols, induced by the 14 alpha-ethyl diol, were prevented by the addition of cholesterol (10 microM or 100 microM) and were reversed by removal of the 14 alpha-ethyl diol from the incubation medium. Incubation of the cells with the 14 alpha-ethyl diol had no effect on the levels of cAMP under the conditions studied. Incubation of the cells with miconazole (10 microM) or with lanosterol (10 microM) was also associated with the accumulation of C30 sterols and an elongation of the cells. 24,25-Dihydrolanosterol (10 microM) also induced similar changes in cellular morphology. The results presented herein demonstrate that marked changes in the sterol composition of CHO-K1 cells can be effected by incubation of the cells with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol, miconazole, or lanosterol. In addition, the findings reported herein indicate an important role of sterols in the control of the shape of these cells.