Cholesterol is a major component of the plasma membrane in mammalian cells, where it acts as a modulator of bulk physical state and integrity. In addition to its structural role, cholesterol is essential for proliferation and other cell processes. The present study was undertaken to explore the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression. Comparisons were made between cholesterol and other sterol analogs that differ from cholesterol in three specific elements: the presence of a Δ⁵ double bond in ring B, the hydroxyl group at C-3, and the presence of an aliphatic side chain. The human leukemia cells HL-60 and MOLT-4 were cultured in cholesterol-free medium and treated with different sterols in the presence or absence of SKF 104976, a competitive inhibitor of lanosterol 14α-demethylase that allows the synthesis of isoprenoid derivatives but not cholesterol. Our results show that the β-hydroxyl group at C-3 and the unsaturated bond at Δ⁵ are necessary for cell proliferation and cell cycle progression. The sterol analog 5α-cholestan-3β-ol (dihydrocholesterol), which is saturated at Δ⁵ and has an A/B ring junction in the trans configuration, was also able to support cell growth. However, 5β-cholestan-3β-ol and 5β-cholestan-3α-ol, both of which have an A/B ring junction in the cis configuration, were totally ineffective in supporting cell growth. Indeed, they produced an inhibition of cell proliferation and arrested the cell cycle specifically in the G2/M phase. These effects of 5β-cholestanols were abrogated by cholesterol in a concentration-dependent manner. Moreover, 5β-cholestanols potently inhibited cholesterol biosynthesis and transcription driven by the sterol response element. In addition to providing a description of the structural features of sterols associated with their supporting action on cell proliferation in mammalian cells, the present results demonstrate that selected cholesterol analogs may act as cytostatic agents, interrupting cell cycle progression specifically in the G2/M phase.