Translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii has been shown previously to require interactions between its 5' untranslated region (5' UTR) and the functions encoded by two nuclear loci, which we name here TBC1 and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC 5' UTR is required for translation initiation. Unlike most procaryotic cis-acting translational control elements, this region has a translational activation function and is located 236 nt upstream from the GUG translation initiation codon. In vivo pulse-labeling of chloroplast-encoded proteins and analyses of the expression of chimeric reporter genes in vivo reveal that a mutation of a newly described locus, TBC3, restores translation from the psbC 5' UTR in the absence of either this cis-acting element or the wild-type trans-acting TBC1 function. These data demonstrate that sequences located in the middle of the psbC 5' UTR, TBC1, and TBC3 functionally interact to control the translation of the psbC mRNA.