The chloroplast protein CSP41a both binds and cleaves RNA, particularly in stem-loops, and has been found associated with ribosomes. A related protein, CSP41b, co-purifies with CSP41a, ribosomes, and the plastid-encoded RNA polymerase. Here we show that Arabidopsis CSP41a and CSP41b interact in vivo, and that a csp41b null mutant becomes depleted of CSP41a in mature leaves, correlating with a pale green phenotype and reduced accumulation of the ATP synthase and cytochrome b ( 6 )/f complexes. RNA gel blot analyses revealed up to four-fold decreases in accumulation for some chloroplast RNAs, which run-on experiments suggested could tentatively be ascribed to decreased transcription. Depletion of both CSP41a and CSP41b triggered a promoter switch whereby atpBE became predominately transcribed from its nucleus-encoded polymerase promoter as opposed to its plastid-encoded polymerase promoter. Together with published proteomic data, this suggests that CSP41a and/or CSP41b enhances transcription by the plastid-encoded polymerase. Gradient analysis of rRNAs in the mutant suggest a defect in polysome assembly or stability, suggesting that CSP41a and/or CSP41b, which are not present in polysomal fractions, stabilize ribosome assembly intermediates. Although psbA and rbcL mRNAs are normally polysome-associated in the mutant, petD-containing RNAs have diminished association, perhaps accounting for reduced accumulation of its respective multimeric complex. In conclusion, our data suggest that CSP41a and CSP41b stimulate both transcription and translation in the chloroplast.