Whereas the biological activities of oxysterols oxidized at C7 (7-ketocholesterol (71(C), 76-hydroxycholesterol (7 6-0HC), 7 alpha-hydroxycholesterol (7 alpha-OHC)) are well documented, those of oxysterols oxidized at C4 (4 beta-hydroxycholesterol (4 beta-OHC), 4 alpha-hydroxycholesterol (4 alpha-OHC)) are not well known, especially on the cells of the central nervous system. Therefore, an improved methodology has been validated for 4 beta-OHC and 4 alpha-OHC synthesis, and the effects on cell viability and cell growth of these molecules were studied on immortalized, tumoral and normal brain cells (158N, C6 and SK-N-BE cells, and mixed primary cultures of astrocytes and oligodendrocytes). Whereas inhibition of cell growth with 7KC, 7 6-OHC, and 7 alpha-OHC is associated with a decrease of cell viability (cytotoxic activities), our data establish that 4 beta-OHC and 4a-OHC have no effect on cell viability, and no or minor effect on cell growth evocating cytostatic properties. Thus, comparatively to oxysterols oxidized at C7, the toxicity of oxysterols oxidized at C4 is in the following range of order: 7KC > 76-OHC > 7 alpha-OHC > (4 alpha-OHC > 4 alpha-OHC). Interestingly, to date, 4 beta-0HC and 4a-OHC are the only oxysterols identified with cytostatic properties suggesting that these molecules, whereas not cytotoxic, may have some interests to counteract cell proliferation.