Cell surface carbohydrate antigens and their metabolism were investigated during the course of differentiation of murine cultured leukemia cells (M1) into macrophage-like cells. The major glycolipids in undifferentiated M1 cells were of the ganglio series, with a small amount of lacto-series glycolipids. A novel branched structure was found as a tetraosylceramide of M1- cells. Upon differentiation, synthesis of lacto-series glycolipids was significantly enhanced and synthesis of globo-series glycolipids was newly induced but the ganglio-series synthesis was much reduced. Undifferentiated cells expressed only i antigen (i+I-Pk-); differentiated macrophage-like cells became I-antigen dominant and Pk-antigen positive (i+/-I+Pk+). The changes proceeded in two sequential steps: (i) an enhancement of lacto-series glycolipid synthesis associated with the conversion of i antigen to I antigen, and (ii) subsequent induction of globo-series glycolipid synthesis accompanied by the appearance of Pk antigen. The experimental system offers a clue for studies on the process of branching (i-to-I conversion) as well as the biological significance of three major glycolipids (globo-, lacto-, and ganglio-series) as markers of cell differentiation.