Identification of transcription initiation sites in the promoter region of the tobacco chloroplast rRNA operon has been carried out by ribonuclease protection of in vitro capped RNAs and primer extension experiments. A promoter with typical chloroplast -10 and -35 motifs (P1) drives initiation of transcription from position -116 relative to the mature 16s rRNA sequence. In addition, we have found that a second primary transcript starts at position -64. This proximal promoter (P2) lacks any elements similar to those reported so far in chloroplast promoter regions, and hence P2 represents a novel-type promoter. Both transcripts are present in chloroplasts from green leaves and in non-photosynthetic proplastids from heterotrophically cultured cells (BY2), but their relative amounts appear to differ. The steady state level of the P2 transcript, with respect to P1, is higher in BY2 proplastids than in leaf chloroplasts.