A form of mammary tumor glycoprotein (MTGP) has been observed to be associated with the sedimentable fraction of homogenates of human carcinomas of the breast. A MTGP-containing breast carcinoma cell line, HS-578T (AY-726), containing both soluble and sedimentable forms of MTGP, was fractionated by differential ultracentrifugation, discontinuous sucrose gradient and on a polyethylene glycol-dextran gradient. The separated crude nuclei fraction, crude mitochondrial fraction, disrupted organelles, ribosomes and rough microsomes, smooth microsomes, and plasmalemma were characterized by electron microscopy and enzymatic markers which confirmed that each fraction was highly enriched in the respective subcellular constituents. Analysis of insoluble MTGP demonstrated significant coisolation with the plasmalemma. On the basis of the present data showing that the sedimentable form of MTGP is firmly associated with the plasmalemma and previous evidence showing the specificity of MTGP for breast carcinoma cells and the surface exposure of MTGP on viable cells, we conclude that sedimentable MTGP is an integral membrane glycoprotein that could have biological significance in the behavior or immunobiology of these tumors.