Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.