The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.