The membrane topology of Escherichia coli lac permease was analyzed using a set of 36 lac permease-alkaline phosphatase (lacY-phoA) gene fusions. The level of enzymatic activity of alkaline phosphatase fused to a cytoplasmic membrane protein appears to reflect whether the fusion junction site normally faces the cytoplasm or periplasm. The alkaline phosphatase activities of cells expressing the lacY-phoA fusions distinguish between models previously proposed for the topology of lac permease and favor one with 12 transmembrane segments. This model is fully compatible with the results of earlier biochemical and immunological studies. The properties of fusions with junctions spanning two of the transmembrane segments at 2- or 3-amino acid intervals indicate that approximately half of the residues of either segment (9-11 amino acids) suffices to promote alkaline phosphatase translocation across the membrane. The additional transmembrane segment amino acids that are not required for this membrane insertion process may normally be needed in unfused lac permease after insertion for stable association with the membrane.