The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques.

In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins.

The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics.

Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria.

Mitochondrial matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship.

The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondrial pool from which they are imported into the mitochondria.