Summary—The effect of extensive differentiation on the synthesis and accumulation of protein 4.1 were studied on Friend erythroleukemia grown in suspension and on fibronectin coated dishes. Whole membranes of Friend erythroleukemia cells (FELC) contained a protein 4.1a and 4.1b doublet ofMr76 and 74 kDa and two minor bands ofMr105 and 43 Da that cross‐reacted with anti‐human protein 4.1 IgG. These proteins were present even in uninduced cells. the synthesis of protein 4.1 was maximal after 4 days of induction in both suspension culture and in fibronectin‐coated dishes whereas the protein 4.1 continued to accumulate until the seventh day. More protein 4.1 accumulated in cells grown on fibronectin‐coated dishes, at each stage of differentiation, than in cells grown in suspension. The protein 4.1a/4.1b ratio changed during differentiation. The amounts of protein 4.1b increased progressively after induction until the protein 4.1a/4.1b ratio was similar to that of mouse mature erythrocyte. The protein 4.1a/4.1b ratio appears to be an internal marker of erythroid differ