Insertion of the alpha- and beta-subunits of amphibian epithelial Na+,K+-ATPase into pancreatic microsomes in cell-free systems was shown to be the same as into membranes of intact cells. The glycoproteic beta-subunit was observed to be cotranslationally inserted into endoplasmic reticulum membranes and to adopt a different pattern of N-linked core and terminal sugars in two different amphibian species. The beta-subunit lacks a cleavable signal sequence but quantitative membrane integration required membrane addition at the start of synthesis. Proteolysis of beta-subunit assembled in vitro indicated a cleavable cytoplasmic domain of about 2000 daltons. The catalytic 98-kilodalton alpha-subunit was also membrane-associated during its synthesis in an alkali-resistant fashion and independent of newly synthesized beta-subunit. In contrast to the beta-subunit, membrane integration of the alpha-subunit was possible as late as a time point in its synthesis which corresponded to about 1/3-1/2 of completion of the nascent chain. A small 34 kDa trypsin-resistant fragment of the alpha-subunit was produced at an early stage of synthesis both in the intact cell and in the cell-free system. These results suggest that membrane insertion of both alpha- and beta-subunit occurs during their synthesis but with a different time course.