A3.01 is a human T cell line previously shown to be defective in cholesterol biosynthesis. Following passage into serum-free medium, A3.01 cells displayed a gradual decline in growth rate which correlated with a depletion of cellular cholesterol content and an accumulation of lanosterol and 24,25-dihydrolanosterol. At the point when cholesterol became undetectable, the growth rate of serum-deprived cells was only one-tenth of the rate observed for serum-supplemented cells. The addition of low density lipoproteins (LDL) restored cellular cholesterol content and resulted in a 7-fold higher growth rate, confirming that cholesterol-deprivation was responsible for the slower growth in the absence of serum. Following prolonged culture in serum-free medium, A3.01 cells underwent a phenotypic change such that the cells achieved a growth rate which was approximately 65% of either LDL-supplemented or cholesterol-proficient cells. This apparent adaptation was not attributable to changes in either fatty acid or sterol composition. These results demonstrate that while cholesterol is preferred, this lymphoid cell line can adapt to the use of lanosterol to satisfy its membrane sterol requirement.