This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12). In addition to identifying residues important for lactose transport function, the analysis revealed that numerous mutations made lac permease highly toxic to cells. In the most extreme cases, production of such proteins at very low steady-state levels reduced cell viability greater than 10(4)-fold. Both frameshift and missense mutations led to toxicity, with the frameshift mutations having the strongest effects observed. The toxic missense mutations corresponded to changes in TM12 expected to interfere with membrane insertion or folding, such as the introduction of charged residues or prolines in the putative helix. The results suggest that cellular toxicity may be a relatively common consequence of mutations altering integral membrane protein folding. An analogous toxicity might contribute to the pathogenesis of several degenerative diseases caused by mutant membrane proteins, such as retinitis pigmentosa, Charcot-Marie-Tooth syndrome, and Alzheimer's disease.