The high-chlorophyll fluorescence photosynthesis mutant hcf109 of Arabidopsis was characterized in detail to gain insights into the regulatory mechanism of RNA processing in higher plants. By using electron transport, chlorophyll fluorescence, and immunoblot studies, we assigned the mutational lesion to photosystems I and II and the plastid NAD(P)H dehydrogenase complex. The functional pleiotropy was reflected in RNA deficiencies. Although all nuclear-encoded photosynthetic RNAs analyzed revealed no difference in size or steady state level between mutant and wild type, the RNA patterns of the plastome-encoded psbB-psbT-psbH-petB-petD, psbD-psbC-ycf9, ndhC-ndhK-ndhJ, and ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndh D transcription units were severely disturbed. These operons encode subunits of photosystems I (psa) and II (psb), the cytochrome bGf complex (pet), the plastid NAD(P)H dehydrogenase (ndh), and the unidentified open reading frame ycf9. With the exception of the ndhC operon, the RNA deficiencies observed were specific and restricted to particular segments of the psbB, psbD/C, and ndhH operons, that is, the psbB-psbT, ycf9, and psaC regions. Run-on transcription studies with isolated chloroplasts showed that the failure of these transcripts to accumulate was due to RNA stability and not transcription. Other polycistronic transcription units analyzed were not affected by the mutation. This result indicates that the trans-regulatory factor encoded by the hcf109 gene is not a general RNA stability factor but that it specifically controls the stability of only these distinct transcripts. Because the hcf109 locus was mapped at a distance < 0.1 centimorgans from the phytochrome C gene, its molecular characterization by positional cloning is possible.