Tim9p is located in the soluble 70-kDa Tim9p-Tim10p complex and the 300-kDa membrane complex in the mitochondrial TIM22 protein import system, which mediates the import of inner membrane proteins. From a collection of temperature-sensitive mutants, we have analyzed two in detail. tim9-3 contained two mutations and tim9-19 contained one mutation, all located near the 'twin CX3C' motif that is conserved in the small Tim proteins. As a result, the import components in the tim9-3 mutant mitochondria were severely reduced and assembled into complexes of aberrant sizes. Protein import was severely reduced and Tim9p and Tim10p binding to in vitro imported ADP/ATP carrier was impaired. In the tim9-19 mutant mitochondria, the 300-kDa membrane complex was assembled, although the soluble 70-kDa Tim9p-Tim10p complex was not detectable. Protein import was decreased only two-fold. When coexpressed in Escherichia coli, tim9-19 and TIM10 proteins failed to assemble into a 70-kDa complex. Our findings suggest that residues near the 'twin CX3C' motif are important for the assembly of Tim9p in both the Tim9p-Tim10p complex and the 300-kDa membrane complex.