Steroid receptor coactivator-1 (SRC-1) specifically bound to the transcription factor NFkappaB subunit p50 but not to p65 as demonstrated by the yeast two hybrid tests and glutathione S-transferase pull down assays. The p50-binding site was localized to a subregion of SRC-1 (amino acids 759-1141) that encompasses the previously described CBP-p300-binding domain. In mammalian cells, SRC-1 potentiated the NFkappaB-mediated transactivations in a dose-dependent manner. Coexpression of p300 further enhanced this SRC-1-potentiated level of transactivations, consistent with the recent findings in which CBP and p300 were shown to be transcription coactivators of the p65 subunit (Perkins, N. D., Felzien, L. K., Betts, J. C., Leung, K., Beach, D. H., and Nabel, G. J. (1997) Science 275, 523-527; Gerritsen, M. E., Williams, A. J., Neish, A. S. , Moore, S., Shi, Y., and Collins, T. (1997) Proc. Acad. Natl. Sci. U. S. A. 94, 2927-2932). These results suggest that at least two distinct coactivator molecules may cooperate to regulate the NFkappaB-dependent transactivations in vivo and SRC-1, originally identified as a coactivator for the nuclear receptors, may constitute a more widely used coactivation complex.