We have recently shown that the IkappaB protein IkappaBbeta interacted with the retinoid X receptor (RXR) and inhibited the 9-cis-retinoic acid (RA)-dependent transactivations (Na, S.-Y., Kim, H.-J., Lee, S.-K., Choi, H.-S., Na, D. S., Lee, M.-O., Chung, M., Moore, D. D., and Lee, J. W. (1998) J. Biol. Chem. 6, 3212-3215). Herein, we show that a distinct IkappaB protein Bcl3 also interacts with RXR, as shown in the yeast two-hybrid tests and glutathione S-transferase pull-down assays. The Bcl3 interaction involved two distinct subregions of RXR, i.e. constitutive interactions of the N-terminal ABC domains and 9-cis-RA-dependent interactions of the C-terminal DEF domains. In contrast to IkappaBbeta, Bcl3 did not interact with the AF2 domain of RXR. Bcl3 specifically interacted with the general transcription factors TFIIB, TBP, and TFIIA but not with TFIIEalpha in the GST pull-down assays. TBP and TFIIA, however, were not able to interact with IkappaBbeta. Accordingly, Bcl3 coactivated the 9-cis-RA-induced transactivations of RXR, in contrast to the inhibitory actions of IkappaBbeta. In addition, coexpression of SRC-1 but not p300 further stimulated the Bcl3-mediated enhancement of the 9-cis-RA-induced transactivations of RXR. These results suggest that distinct IkappaB proteins differentially modulate the 9-cis-RA-induced transactivations of RXR in vivo.