The regulation of gene expression is still one of the major issues in modern plant molecular biology. The amount of RNA in a cell is regulated by both transcriptional and posttranscriptional events. Methods to determine these steady-state levels of RNAs, such as Northern analysis, ribonuclease protection assay (RPA), and quantitative real-time PCR, do not discriminate between regulation by de novo RNA synthesis and the influence by degradation or stabilization. To assess the rate of transcription of individual genes, run-on transcription is utilized. To this end, isolated chloroplasts are used in brief in vitro transcription reactions in the presence of radiolabeled nucleotides, with a subsequent hybridization of the isolated RNA with DNA fragments spotted on membranes. Here, we describe a protocol for run-on transcription in chloroplasts isolated from Arabidopsis leaves and present data on the transcriptional activity of several plastid genes in detached leaves of different Arabidopsis ecotypes.