An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for beta-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyl-transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.