Energy coupling factor (ECF) transporters take up micronutrients in Bacteria and Archaea. They consist of a membrane-embedded S-component that provides substrate specificity and a three-subunit ECF module that couples ATP hydrolysis to transport. The S-components ThiT (for thiamin) and NiaX (for niacin) from Lactococcus lactis form complexes with the same ECF module. Here, we assayed the uptake of thiamin and niacin in Escherichia coli cells expressing the transporter genes. We demonstrate that the two different S-components compete for the ECF module, and that competition is more efficient in the presence of the transported substrate. The data suggest that binding and release of the S-components is a step in the transport cycle.