17beta-Estradiol (E2)-induced rapid functions (from seconds to minutes) can be attributed to a fraction of nuclear estrogen receptor-alpha (ERalpha) localized at the plasma membrane. As a potential mechanism, we postulated that S-palmitoylation of the Cys447 residue may explain the ability of ERalpha to associate to plasma membrane making possible E2-dependent rapid functions [e.g., extracellular regulated kinase (ERK) activation]. Here, we report direct evidence that the mutation of the Cys447 residue to Ala impairs human ERalpha palmitoylation and E2-induced rapid ERK phosphorylation when transfected in ER-devoid HeLa cells. Moreover, the Cys447Ala mutation significantly decreases the E2-induced transactivation of an estrogen responsive element construct probe. Similar effects were obtained treating HeLa cells transfected with wild type ERalpha with the palmitoyl-acyltransferase inhibitor 2-bromo-hexadecanoic acid. Moreover, the deletion of the A-D domains (containing the DNA binding region) of ERalpha had no consequences on [(3)H]palmitate incorporation, whereas no palmitoylation occurred in the ERalpha mutant devoid of the E domain (i.e., ligand binding domain). These results point to the pivotal role of the Cys447 residue in ERalpha palmitoylation and in the modulation of E2-induced non-genomic functions.