In general, in higher plants, the core subunits of a bacterial-type plastid-encoded RNA polymerase (PEP) are encoded by the plastid rpoA, rpoB, rpoC1 and rpoC2 genes. However, an rpoA gene is absent from the moss Physcomitrella patens plastid genome, although the PpRpoA gene (renamed PpRpoA1) nuclear counterpart is present in the nuclear genome. In this study, we identified and characterized a second gene encoding the plastid-targeting α subunit (PpRpoA2). PpRpoA2 comprised 525 amino acids and showed 59% amino acid identity with PpRpoA1. Two PpRpoA proteins were present in the PEP active fractions separated from the moss chloroplast lysate, confirming that both proteins are α subunits of PEP. Northern blot analysis showed that PpRpoA2 was highly expressed in the light, but not in the dark, whereas PpRpoA1 was constitutively expressed. Disruption of the PpRpoA1 gene resulted in an increase in the PpRpoA2 transcript level, but most plastid gene transcript levels were not significantly altered. This indicates that transcription of most plastid genes depends on PpRpoA2-PEP rather than on PpRpoA1-PEP. In contrast, the transcript levels of petN, psbZ and ycf3 were altered in the PpRpoA1 gene disruptant, suggesting that these are PpRpoA1-PEP-dependent genes. These observations suggest that plastid genes are differentially transcribed by distinct PEP enzymes with either PpRpoA1 or PpRpoA2.