The translocase of the inner membrane 17 (AtTIM17-2) protein from Arabidopsis has been shown to link the outer and inner mitochondrial membranes. This was demonstrated by several approaches: (i) In vitro organelle import assays indicated the imported AtTIM17-2 protein remained protease accessible in the outer membrane when inserted into the inner membrane. (ii) N-terminal and C-terminal tagging indicated that it was the C-terminal region that was located in the outer membrane. (iii) Antibodies raised to the C-terminal 100 amino acids recognize a 31-kDa protein from purified mitochondria, but cross-reactivity was abolished when mitochondria were protease-treated to remove outer membrane-exposed proteins. Antibodies to AtTIM17-2 inhibited import of proteins via the general import pathway into outer membrane-ruptured mitochondria, but did not inhibit protein import via the carrier import pathway. Together these results indicate that the C-terminal region of AtTIM17-2 is exposed on the outer surface of the outer membrane, and the C-terminal region is essential for protein import into mitochondria.