Biochemical analyses of protein translocation intermediates formed during early stages of protein import into chloroplasts under restricted energy conditions identified many components involved in protein translocation. Whereas limited information is available at the latter stages of import, as it is difficult to pause the movement of precursor once it was released from the early intermediates. To address this problem, we have attempted to obtain post-early intermediates to plug translocation channel by precursor proteins carrying a tightly-folded structure at their C-termini. To this end, we prepared the recombinant precursor protein whose chloroplastic targeting signal was fused to dihydrofolate reductase from Escherichia coli, known to fold tightly in the presence of its substrate analogue methotrexate. If the precursor was treated with methotrexate prior to the import reaction, the amount of processed precursor was reduced. However, the processed precursor was recovered in the soluble fraction after fractionation, indicating that methotrexate was released from the precursor, which suggested the presence of strong unfolding activity within chloroplasts.