The lipid requirements of a human macrophagelike cell line were studied. The cells grew only about one generation in a medium supplemented with delipidated serum; during the growth the cholesterol content of the cells was depleted. Growth was restored by including in the medium serum lipids subjected to alkaline hydrolysis or cholesterol. The extent of growth was dependent on cholesterol concentration. No growth was obtained with 5‐cholestene, 5‐cholesten‐3‐one, cholesteryl chloride, coprostanol, β‐sitosterol, orstigmasterol. Very limited growth occurred with cholesterol methylether, epicholesterol, or β‐cholestarol. Therefore, for optimal growth of these cells there is a stringent requirement for the structural features of cholesterol, which include a 3‐βOH group, a Δ⁵ ‐double bond, a trans ring A/B configuration, and freedom of the side chain from bulky groups. This stringency far exceeds what was previously reported for other cells. Of the six sterols that failed to support growth at all, five were incorporated into cells moderately to extensively. This suggests that assembly of a functional membrane is impaired when these sterols are used as substrates for growth.