The chloroplast S10 ribosomal protein operon is partially duplicated in many plants because it initiates within the inverted repeat of the circular chloroplast genome. In spinach, the complete S10 operon (S10B) spans the junction between inverted repeat B (IRB) and the large single-copy (LSC) region. The S10 operon is partially duplicated in the inverted repeat A (IRA), but the sequence of S10A completely diverges from S10B at the junction of S10A and the LSC region. The DNA sequence shared by S10A and S10B includes trnI1, the rpl23 pseudogene (rpl23 psi), the intron-containing rpl2 and rps19, which is truncated in S10A at the S10A/LSC junction (rps19'). Transcription of rps19' from the promoter region of S10A could result in the synthesis of a mutant S19 protein. Analysis of RNA accumulation and run-on transcription from S10A and S10B using unique probes from the S10A/LSC and S10B/LSC junctions reveals that expression of S10A is reduced. The difference in S10A and S10B expression appears to be the result of reduced transcription from S10A, rather than differences in RNA stability. Transcription of S10B can initiate at three distinct promoter regions, P1, P2 and P3, which map closely to transcripts detected by S1 nuclease analysis. P1 is located upstream of trnI1 and has the highest transcription initiation frequency in vitro of the three promoter regions. The DNA sequence of P1 is most similar to the chloroplast promoter consensus DNA sequence. Interference by the highly and convergently transcribed psbA-trnH1 operon is considered as a mechanism to explain the reduced activity of the S10A promoters.