In this study, we identified a lowluminescence mutant referred to as rll4 (reduced LUC luminescence 4) by screening a M₂ population derived from an ethyl methane sulfonate (EMS)mutagenized transgenic parental line ColLUC, which harbors a firefly luciferase reporter (LUC) gene under the control of 2×35S promoter. Genetic analysis demonstrated that the rll4 locus contains a single nuclearencoded recessive mutation. By using mapbased cloning strategy, we narrowed down the rll4 locus between the molecular markers CL417B10M1 and CL418B2M2, which are located at BAC (Bacterial Artificial Chromosome) clones F20D10 and F20M13 on chromosome 4, respectively. Methylationsensitive restriction enzyme PCR (ChopPCR) results showed the DNA methylation levels of a few genomic loci were increased in rll4 mutant. Reverse transcriptionPCR (RTPCR) results revealed that the expression levels of several endogenous targets of RNAdirected DNA methylation (RdDM) pathway were decreased in rll4 mutant while the expression of ROS1 (REPRESSOR OF SILENCING 1) remained unchanged. Taken together, these results suggest that RLL4 locus is probably involved in DNA demethylation process in A. thaliana.