Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, is anchored to the membrane by two transmembrane domains at the two ends of the molecule. The transmembrane domains are important for enzymatic activity, as mutants lacking one or both of these domains have a fraction of the enzymatic activity of the wild-type CD39. We investigated the interactions between the transmembrane domains by using a strain of yeast that requires surface expression of CD39 for growth. Random mutagenesis of selected amino acid residues in the N-terminal transmembrane domain revealed that the presence of charged amino acids at these positions prevents expression of functional protein. Rescue of the growth of these mutants by complementary mutations on selected residues of the C-terminal transmembrane domain indicates that there is contact between particular faces of the transmembrane domains.