Proteins unique to B16 melanoma cells grown in culture and as solid tumors have been independently described recently. As both proteins have apparent M(r) of 65,000-70,000, we have more fully compared their biochemical characteristics. The M(r) 65,000 protein, which is isolated from B16 cells in culture, has an aggregate molecular weight of >200,000 under nonreducing conditions. This M(r) 65,000 form is shed into the culture medium exclusively and is the predominant molecular species of serum-free culture supernatants. It was found that B700, a major membrane protein of B16 cells in solid tumors, and the M(r) 65,000 preparation from cultured cells were electrophoretically identical when compared under four separate sets of conditions. These two proteins also share immunologic determinants. The results suggest the identity of these two proteins isolated by completely different approaches.