Plastidic type I signal peptidase 1 (Plsp1) is necessary for proper chloroplast development although its mode of action is not completely understood. Here, we demonstrate by immunoblotting and electron microscopy immunolocalization that Plsp1 is evenly distributed in the envelope and thylakoids of developing chloroplasts in meristems, whereas it is mainly located in thylakoids of developed chloroplasts in leaf mesophyll. This localization pattern corresponded to accumulation of transcripts for Plsp1 substrates in the envelope and thylakoids, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) and oxygen-evolving complex subunit 33 (OE33), respectively. Furthermore, developing chloroplasts processed Toc75 more efficiently than did developed chloroplasts. These findings help establish the rationale for multiple localizations of Plsp1.