The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.