Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N–terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid-transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling-lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1-null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid-transfer signal removal on protein targeting and accumulation by examining the localization of non-mature lumenal proteins in the Arabidopsis plsp1-null mutant and performing a protein import assay using pea chloroplasts. In plsp1-null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non-mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid-transfer signal removal did not disrupt cpSEC- and cpTAT-dependent translocation, but prevented release of proteins from the membrane. Interestingly, non-mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440-kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1-null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.