Human embryonic kidney (K293) cells transfected with a mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc) were utilized to investigate the potential effects of trans-retinoic acid (tRA), either by itself or in combination with glucocorticoid (GC) hormones, on a well-characterized, GC-sensitive transcriptional response. tRA or the synthetic GC hormone dexamethasone induced transcription from the MMTV promoter in a dose-dependent manner, with 1 micromol tRA and 1 micromol dexamethasone alone causing a four- to six-fold and a 40-fold induction of basal transcription, respectively. Simultaneous treatment with 1 micromol dexamethasone and 1 micromol tRA resulted in a synergistic transcriptional response that was 120-fold higher than basal level and 2.5 times the predicted response, based on a simple additive effect of both agonists. tRA does not appear to mediate this synergistic transcriptional response by enhancing GC receptor (GR) binding capacity, affinity, or nuclear translocation. tRA was unable to potentiate GC-induced transcriptional activity from a minimal GC response element (GRE), and GC were unable to potentiate tRA-induced transcriptional activity from a minimal retinoic acid response element (RARE). These data rule out direct protein-protein interactions between GC and retinoid receptors as a mechanism for the observed synergism. tRA also synergized with aldosterone-induced, mineralocorticoid receptor (MR)-mediated, transcriptional activation of the MMTV promoter, resulting in a response that was 1.7 times the predicted additive response. The MMTV GRE located between -187 and -165 was required for GC-induced and synergistic activation of the MMTV promoter, whereas sequences located within -151 to +5 were sufficient for tRA-induced transcription from the MMTV promoter. Mutation of a consensus RARE half-site (CCAAGT) identified at position -65 to -60 within the MMTV-LTR did not affect either tRA-induced transcriptional activation or synergism with GC. We propose that the tRA-induced transcriptional response from the MMTV promoter, as well as synergism with GC, may be mediated by the activation or induction of a factor(s) that either directly binds to the MMTV promoter or indirectly stabilizes binding of another transcription factor to these sequences.