The 3' maturation of chloroplast pre-mRNAs in Chlamydomonas proceeds via endonucleolytic cleavage, exonucleolytic trimming of the upstream cleavage product, and rapid degradation of the downstream moiety. However, the cis elements and trans factors remain to be characterized in detail. In the case of atpB, a 300 nucleotide processing determinant (PD), consisting of an inverted repeat (IR) and endonuclease cleavage site (ECS), directs 3' maturation. To further characterize the PD, 15 variants were examined in vivo in ectopic contexts. This revealed that the IR, and nucleotides 15-37 downstream of the ECS stimulate processing. A candidate trans factor for 3' maturation was subsequently functionally analyzed. This factor is encoded by the nuclear locus MCD4, and the mcd4 mutant was known to accumulate abnormally 3'-processed chloroplast mRNAs. When the mcd4 mutation was crossed into strains containing reporter genes with insertions of several PD versions, processing was reduced in some cases. This caused accumulation of RNA sequences downstream of the PD, which are normally degraded. From these data, it can be suggested that MCD4 facilitates the endonucleolytic cleavage step in 3' end maturation of atpB and perhaps other mRNAs, by interacting with the IR, RNA downstream of the IR, or with proteins bound there.