We have shown that four metabolites of all-trans-retinoic acid (ATRA) (4-oxo-, 4-OH-, 18-OH-, and 5,6-epoxy-RA) can induce maturation of NB4 promyelocytic leukemia cells (Idres, N., Benoit, G., Flexor, M. A., Lanotte, M., and Chabot, G. G. (2001) Cancer Res. 61, 700-705). To better understand the mechanism of action of ATRA metabolites and isomers, we assessed their binding to retinoic acid receptors (RARs) and activation of RAR-mediated transcription via a retinoic acid response element (RARE). Competition binding experiments with tritiated ATRA showed that all metabolites could bind to RARs with variable affinity. For transactivation studies, COS-7 cells were cotransfected with RAR alpha, beta, or gamma expression vectors and the reporter plasmid RARE-tk-Luc, and the retinoid concentrations for half-maximal luciferase activity (EC(50)) were determined. All retinoids tested could activate the three RAR isotypes. The lowest EC(50) value for RAR alpha was with 9-cis-RA (13 nM), followed by 4-oxo-RA (33 nM), 5,6-epoxy-RA (77 nM), 13-cis-RA (124 nM), 18-OH-RA (162 nM), ATRA (169 nM), and 4-OH-RA (791 nM). For RAR beta, the EC(50) values increased as follows: 4-oxo-RA (8 nM), ATRA (9 nM), 18-OH-RA (14 nM), 5,6-epoxy-RA (35 nM), 13-cis-RA (47 nM), 4-OH-RA (64 nM), and 9-cis-RA (173 nM). For RAR gamma the EC(50) values were: ATRA (2 nM), 5,6-epoxy-RA (4 nM), 18-OH-RA (14 nM), 13-cis-RA (36 nM), 9-cis-RA (58 nM), 4-oxo-RA (89 nM), and 4-OH-RA (94 nM). By comparing the -fold induction of luciferase activity, all retinoids tested were equipotent at transactivating RARE-tk-Luc whatever the RAR considered. However, the best induction of the transcription was obtained for RAR alpha, which was 5-fold higher than for RAR beta and 10-fold higher than for RAR gamma. In conclusion, these data show that ATRA metabolites can bind to and activate the three RARs with variable relative affinity but with similar efficacy. These results suggest that ATRA metabolites may activate several signaling pathways and probably play an important role in cellular physiology and cancer therapy.