Pit2 is a member of the Pit family of inorganic phosphate transporters and serves as a gamma-retrovirus receptor in mammals. Pit2 contains two copies of the protein homology domain PD001131, which defines the Pit family. These domains are presumably in opposite topology with respect to the plasma membrane plane. We have mutated a serine residue conserved in almost all of the 192 known PD001131 sequences to alanine in each PD001131 domain of human Pit2. Expression in CHO cells showed that phosphate uptake was affected severely in mutants, whereas susceptibility to virus infection was conserved. We reported previously that the inorganic phosphate concentration affects both phosphate transport mediated by Pit2 and the conformation of cell-surface Pit2 oligomers. Cross-linking experiments in transport-incompetent Pit2 mutants indicated that structural changes induced by phosphate starvation or supply occur independently of the whole transport cycle. These results suggest that the structural reorganisation of cell-surface Pit2 occurred as a consequence of ion binding, a model consistent with the possible involvement of cell-surface Pit2 oligomers in inorganic phosphate sensing.