In the brain, mineralocorticoid (MR) and glucocorticoid receptors (GR) act as transcription factors to mediate effects of glucocorticoid (GC) hormones in system homeostasis and stress adaptation. The MR and GR have a high degree of homology in their DNA binding (DBD) and ligand binding domain (LBD), but differ considerably in the N-terminal domain (NTD). MR and GR mediate different effects of GCs, but also share target genes. Here we assessed interactions between MR and GR in murine neuroblastoma cells treated with corticosterone. Using a proximity ligation assay, we described the role of the DBD, LBD and NTD in cell nuclear MR and GR interactions. Using FKBP5 mRNA as a readout we defined conditions in which transactivation depends on MR:GR heterodimers, using DBD ‘salt bridge mutants’. Our data provide a steppingstone to study intranuclear behavior of the receptors, and to characterize the transcriptome of MR:GR heterodimers. These data may help develop pharmacological interventions for a diverse range of psychiatric disorders that could improve cognitive and emotional function.