We show that mice expressing retinoid X receptor beta (RXRbeta) impaired in its transcriptional activation function AF-2 (Rxrb(af20) mutation) do not display the spermatid release defects observed in RXRbeta-null mutants, indicating that the role of RXRbeta in spermatid release is ligand-independent. In contrast, like RXRbeta-null mutants, Rxrb(af20) mice accumulate cholesteryl esters in Sertoli cells (SCs) due to reduced ABCA1 transporter-mediated cholesterol efflux. We provide genetic and molecular evidence that cholesterol homeostasis in SCs does not require PPARalpha and beta, but depends upon the TIF2 coactivator and RXRbeta/LXRbeta heterodimers, in which RXRbeta AF-2 is transcriptionally active. Our results also indicate that RXRbeta may be activated by a ligand distinct from 9-cis retinoic acid.