The maltose transporter of Escherichia coli is a hetero-oligomeric complex located in the cytoplasmic membrane of the cell. The in vivo assembly of this complex has been examined by using an assay based on the proteolytic sensitivity of one of its components, MalF. Immediately after synthesis and insertion into the membrane, MalF is sensitive to exogenously added proteases. In a time- and complex assembly-dependent fashion, MalF becomes protease resistant. Using this assay, we show that MalF is inserted into the membrane independently of other components of the transport complex. The assembly of the maltose transport complex occurs subsequently from a pool of freely diffusing protein in the membrane. This assembly process is efficient and occurs with rapid kinetics.