The major protein composition and secretory character of rough microsomes isolated from the C3HBA mammary adenocarcinoma have been determined for the tumor borne in virgin female and lactating mice. Rough microsomes isolated from tumors have an equilibrium density distribution in a linear 1.0 to 2.0 M sucrose gradient similar to that exhibited by rough microsomes isolated from normal epithelium during lactation. Fractionation of the rough microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that, although the molecular weights of the major proteins are the same in normal and neoplastic rough microsomes, the relative levels at which these major proteins occur in neoplastic rough microsomes are distinct from those observed in normal rough microsomes isolated at various stages of differentiation. The most significant change in protein composition following neoplastic transformation is the presence of a high level of protein with a molecular weight of 200,000. There are changes in the relative levels of the major proteins of the rough endoplasmic reticulum attending transplantation of the tumor from virgin female to lactating mice; these changes, however, are quite different from those that occur in the rough endoplasmic reticulum of normal epithelium at the time of parturition. Rough microsomes isolated from tumors borne in virgin female mice discharge more than 90% of their nascent polypeptide chains extravesicularly upon premature termination by puromycin; there is no change in the vectorial discharge of these puromycyl peptides following implantation of the tumor into lactating mice. Thus, by a functional criterion, the rough endoplasmic reticulum of mammary adenocarcinoma cells appears similar to that found in normal, nonsecretory epithelium.