Control of gene expression in chloroplasts is critically dependent upon post-transcriptional mechanisms, most of which require formation of RNA-protein complexes. The 5' untranslated regions (5'UTRs) of chloroplast mRNAs have been shown to affect stability and/or translation of the message. These effects are mediated by the binding of specific protein(s) to the 5'UTR. We can detect such 5'UTR-protein complexes in vitro and have previously shown that the same polypeptide(s) bind many spinach chloroplast 5'UTRs (Robida et al. 2002). Here we report investigations on the RNA elements and protein factors involved in formation of these complexes. Comparison of the atpI 5'UTR, which serves as the representative 5'UTR for these experiments, among 12 angiosperms revealed two phylogenetically conserved regions upstream of a putative ribosome binding site. To determine whether the two conserved regions interact to form a single polypeptide-binding site, binding assays were performed with RNAs containing only one of the two. Those experiments revealed that the entire 5'UTR could be separated into two binding sites for chloroplast polypeptides, each containing one of the two conserved regions. Competition binding assays using the individual binding sites established that each was bound by different polypeptide(s). These data support the hypothesis that there are at least two unique polypeptides involved in these 5'UTR-protein complexes, each binding specifically to a different site within the 5'UTR.