The Arabidopsis mutant heat-intolerant 4-1 (hit4-1) was isolated from an ethyl methanesulphonate-mutagenized M2 population on the basis of its inability to withstand prolonged heat stress (4 days at 37°C). Further characterization indicated that hit4-1 was impaired specifically in terms of basal but not acquired thermotolerance. Map-based cloning revealed that the HIT4 gene encoded a plant-specific protein for which the molecular function has yet to be studied. To investigate the cellular role of HIT4 and hence elucidate better its protective function in heat tolerance in plants, a GFP-HIT4 reporter construct was created for a protoplast transient expression assay. Results showed that fluorescently tagged HIT4 was localized to the chromocentre, a condensed heterochromatin domain that harbours repetitive elements for which transcription is normally suppressed by transcriptional gene silencing (TGS). DAPI-staining analysis and FISH with a probe that targeted centromeric repeats showed that heat-induced chromocentre decondensation was inhibited in nuclei of hit4-1 subjected to direct heat treatment, but not in those that were allowed to acquire thermotolerance. Moreover, heat reactivation of various TGS loci, regardless of whether they were endogenous or transgenic, or existed as a single copy or as repeats, was found to be attenuated in hit4-1. Meanwhile, the levels of transcripts of heat shock protein genes in response to heat stress were similar in both hit4-1 and wild-type plants. Collectively, these results demonstrated that HIT4 defines a new TGS regulator that acts at the level of heterochromatin organization and is essential for basal thermotolerance in plants.