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Sterol homeostasis in eukaryotic cells relies on the§reciprocal interconversion of free sterols and steryl§esters. Excess free sterols are esterified to steryl§esters, the storage form of sterols, by§acyltransferases. Upon cellular demand, steryl esters§are hydrolyzed to free sterols and fatty acids by§three sterol ester hydrolases. We have identified a§novel sterol modification in yeast, the§sterol acetylation/deacetylation cycle. Sterol§acetylation requires the acetyltransferase, ATF2,§whereas deacetylation requires a membrane-anchored§deacetylase, SAY1. Lack of SAY1 results in the§secretion of acetylated sterols into the culture§medium. Acetylation and export of§the steroid hormone precursor pregnenolone depends on§its acetylation by ATF2, but is independent of§SAY1-mediated deacetylation, indicating that the§substrate specificity of the deacetylase determines§whether acetylated sterols and steroids are secreted§from the cells or whether they are deacetylated and§retained. Human AADAC (aryl acetamide deacetylase)§complements Say1 activity and is thus a functional§homologue of SAY1. These findings indicate that at§least a part of this pathway is evolutionarily§conserved.