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We have isolated five members of the multigene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase in petunia and examined their expression in petunia leaves. Of the five rbcS genes, two (ssu11A and ssu8) are expressed at high levels in petunia leaves. Northern analysis using gene specific oligonucleotide probes revealed that ssu11A accounts for 40% of the total rbcS transcripts in petunia leaves while ssu8 accounts for 4 to 5% of the total rbcS transcripts. Structural comparisons of ssu8 and ssu11A revealed that the coding sequence of ssu8 is interrupted by three introns, while the coding sequence of ssu11A is interrupted by two introns. The positions of the first two introns are identical, the third intron in ssu8 is located in a highly conserved region of the protein. The 5' and 3' flanking sequences of ssu11A are highly homologous to the 5' and 3' flanking sequences of ssu8. S1 nuclease mapping was used to locate the start of transcription of ssu8 and ssu11A and showed that ssu8 mRNA leader differs in sequence from the ssu11A mRNA leader.