The expression of arginine-vasopressin (AVP) is regulated by estradiol and testosterone (T) in different neuronal populations by mechanisms that are not yet fully understood. Estrogen receptors (ERs) have been shown to participate in the regulation of AVP neurons by estradiol. In addition, there is evidence of the participation of ERβ in the regulation of AVP expression exerted by T via its metabolite 5α-dihydrotestosterone (5α-DHT) and its further conversion in the androgen metabolite and ERβ ligand 3β-diol. In this study we have explored the role of ERs in the regulation exerted by estradiol and T on AVP expression, using the human neuroblastoma cell line SH-SY5Y. Estradiol treatment increased AVP mRNA levels in SH-SY5Y cells in comparison with cells treated with vehicle. The stimulatory effect of estradiol on AVP expression was imitated by the ERα agonist 4,4′,4′,-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and blocked by the ER antagonist, ICI 182,780, and the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile reduced AVP expression, whereas the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol enhanced the action of estradiol on AVP expression. T increased AVP expression in SH-SY5Y cells by a mechanism that was dependent on aromatase but not on 5α-reductase activity. The T effect was not affected by blocking the androgen receptor, was not imitated by the T metabolite 5α-DHT, and was blocked by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, 5α-DHT had a similar effect as the ERβ agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and 3β-diol, reducing AVP expression. These findings suggest that estradiol and T regulate AVP expression in SH-SY5Y cells through ERs, exerting a stimulatory action via ERα and an inhibitory action via ERβ.