Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus TRbetaA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte nuclei, and eliminates regulation of transcription by TR-RXR. Expression of Xenopus RPD3p, the catalytic subunit of histone deacetylase, represses the TRbetaA promoter, but only after efficient assembly of the template into nucleosomes. In contrast, the unliganded TR-RXR represses templates only partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression by the deacetylase alone, compared with those needed in the presence of unliganded TR-RXR. In addition, whereas hormone-bound TR-RXR targets chromatin disruption as assayed through changes in minichromosome topology and loss of a regular nucleosomal ladder on micrococcal nuclease digestion, addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR-RXR can facilitate transcriptional repression in the absence of hormone through mechanisms in addition to recruitment of deacetylase, and disrupts chromatin structure through mechanisms in addition to the inhibition or release of deacetylase.