The plastid gene rpoB encodes a plastid‐specific, DNA‐dependent RNA polymerase. A study of a barley mutant, albostrians, indicated that rpoB itself is transcribed by a nuclear‐encoded RNA polymerase. However, the molecular nature of the nuclear‐encoded RNA polymerase and mechanisms of the transcriptional regulation of rpoB expression have not been elucidated. Using an in vivo transient assay system for gene expression in plastids, the 5′‐flanking region of rpoB from Arabidopsis thaliana in plastids of cultured BY‐2 tobacco cells was analyzed. A series of deletion analyses of the 5′‐flanking region indicated that there were two positive and one negative regulatory regions for rpoB expression. Comparison of the 5′‐flanking sequences of rpoB among several plant species revealed the existence of three conserved motifs in these regions.