Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.