Although missense mutations that inactivate integral membrane proteins cause a variety of diseases, the mechanisms by which they act are poorly understood. To establish a model for investigating this issue, we identified 51 missense mutations arising in vivo that inactivate Escherichia coli lac permease, a well-characterized membrane transport protein. The mutants were isolated using a genetic screening procedure which eliminates mutations that block expression of the lac permease gene, such as nonsense and frameshift mutations. The majority of the 51 missense mutations caused highly non-conservative changes in membrane-spanning sequences, such as the introduction of charged residues. Nevertheless, the greatest clustering of substitutions occurred in the two regions of lac permease thought to be most important for transport function. The existence of this clustering indicates that even highly non-conservative substitutions may cause relatively localized structural defects. Conservative inactivating substitutions were scattered throughout lac permease and may affect residues that make contacts required for normal folding. Two unexpected phenotypes were observed in the collection of mutants: about 20% of the substitutions led to cold-sensitive lactose utilization, and one substitution made the mutant lac permease toxic to cells. This relatively unbiased collection of mutants should provide a resource for further studies of how missense mutations inactivate membrane proteins in vivo.