In many developmentally and functionally important higher plant plastid genes, expression depends on a specific nuclear-encoded RNA polymerase (NEP). Molecular mechanisms for NEP-mediated gene expression are poorly understood. We have improved a transient expression assay based on biolistics and the dual-luciferase reporter technique, which facilitated investigations into the regulation of plastid genes in vivo. We scrutinized the 5'-flanking region and the 5'-untranslated region (5'UTR) of accD, a plastid gene encoding a subunit of the prokaryotic-type acetyl-CoA carboxylase which is transcribed exclusively by NEP. The results indicated that two AT-rich sequences, one of them containing two overlapping YRTA-like motifs, were essential for accD expression in vivo. The results also revealed that the length of the 5'UTR rather than a particular sequence element was a determinant for the level of accD expression. Because transcripts accumulated in proportion to reporter enzyme activity and protein levels, and transcript degradation rates were independent of the nature of the 5'UTR, it was unlikely that the 5'UTR acts as a translational enhancer or a stabilizer of the transcripts. Therefore, the length of 5'UTR might be a factor contributing to the efficiency of NEP-dependent transcription in plastids.