Protein import into chloroplasts is an energy-requiring process mediated by a proteinaceous import apparatus. Although previous work has shown that low levels of ATP or GTP can support precursor binding, the role of GTP during the import process remains unclear. Specifically, it is unknown whether GTP plays a separate role from ATP during the early stages of protein import and whether GTP has any role in the later stages of transport. We investigated the role of GTP during the various stages of protein import into chloroplasts by using purified GTP analogs and an in vitro import assay. GTP, GDP, the nonhydrolyzable analog GMP-PNP, and the slowly hydrolyzable analogs guanosine 5'-O-(2-thiodiphosphate) and guanosine 5'-O-(3-thiotriphosphate) were used in this study. Chromatographically purified 5'-guanylyl-imido-diphosphate and guanosine 5'-O-(3-thiotriphosphate) were found to inhibit the formation of early-import intermediates, even in the presence of ATP. We also observed that GTP does not play a role during the translocation of precursors from the intermediate state. We conclude that GTP hydrolysis influences events leading to the formation of early-import intermediates, but not subsequent steps such as precursor translocation.