In this study, the homodimeric and heteromeric nature of ProP, an H+/solute symporter, from E. coli was analyzed. The measured initial rates of proline uptake via ProP-His6 and His6-ProP indicated that as the growth medium osmolality increased, the assay medium osmolality required for half maximal transport activity (Π½/RT) increased and the maximal uptake rate (Amax) decreased. The oligomeric state of ProP, as determined by Blue Native PAGE, showed that both monomeric and dimeric forms of the transporter were present in wild type and cardiolipin deficient bacteria expressing ProP, ProP-His6 or His6-ProP, after culturing in low or high growth medium osmolality. The BACTH System was used to confirm the homodimeric ProP-ProP interaction and to verify the heteromeric interaction between ProP and YdhP. Initial rates of proline uptake via ProP and Western blots indicated that replacement of the ydhP locus with a kanamycin cassette had no effect on ProP function or expression.