To evaluate the contribution of ERK1/2 phosphorylation of estrogen receptor (ER) to activation and repression of endogenous genes, we produced stably transfected lines of HeLa cells with functional ERK1/2 pathways that express similar levels of wild-type human ER and ER mutated to inactivate the well-known Map kinase site at Serine 118 (ERS118A). We compared effects of the S118A mutation on 17-estradiol (E₂)-mediated transactivation, which is heavily dependent on activation function 2 (AF2) of ER and on 4-hydroxytamoxifen (OHT)-mediated transactivation, which is heavily dependent on AF1, which includes S118. To examine whether S118 was the key ERK/Map kinase phosphorylation site in ER action, we compared the effects of the S118A mutant and the ERK inhibitor U0126 on expression of endogenous genes. In several estrogen response element (ERE)-containing genes the S118A mutation strongly reduced induction by E₂ and U0126 did not further reduce expression. Expression of another group of ERE-containing genes, was largely unaffected by the S118A mutation. The S118A mutation had variable effects on genes induced by ER tethering or binding near SP1 and AP-1 sites. For 5 mRNAs whose expression is strongly down-regulated by E₂ and partially or completely down-regulated by OHT, the S118A mutation reduced or abolished down-regulation by E₂ and nearly abolished down-regulation by OHT. In contrast, for SMAD3, which is down-regulated by E₂ and not by OHT, the S118A mutation had little effect. These data suggest that there may be distinct groups of genes down-regulated by ER and suggest a novel role for ERK phosphorylation at serine 118 in AF1 in regulating expression of the set of genes down-regulated by OHT.