The effect of 17beta-estradiol (E2) on the proliferation of cardiac fibroblasts (CFs) remains controversial. This study investigated which subtype of estrogen receptor (ER), ERalpha or ERbeta, mediated the effect of E2 on CF growth by the gain of function analysis using an adenovirus vector. One hundred nanomoles per liter of E2 attenuated DNA synthesis by up to 10%, and transactivated the estrogen-responsive element determined by luciferase assay in rat neonatal CFs. We constructed replication-deficient adenoviruses bearing the coding region of human ERalpha, ERbeta, or the dominant-negative form of ERbeta (designated AxCAERalpha, AxCAERbeta, and AxCADNERbeta, respectively). When Us were infected with AxCAERalpha or AxCAERbeta at multiplicity of infection of 20 or higher, DNA synthesis was decreased by 50% in response to E2 and the effect was abolished by co-infection with AxCADNERbeta. Similarly, transcriptional activity of ER in CFs infected with AxCAERalpha or AxCAERbeta was markedly enhanced and co-infection With AxCADNERbeta abolished the effects. These results suggest that E2 inhibits CF growth and that both ER subtypes mediate the effect comparably and redundantly. (C) 2003 Elsevier Inc. All rights reserved.