A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.