We conducted a study on autoinduction of differentiation in human myelocytic leukemia cells (HL-60-Y3) in which the effects of serum cytodifferentiation were excluded by the use of a serum-free semisolid culture. In the culture dish the HL-60-Y3 colony count per dish was kept at 100 or below, and only the formation of clumping-type colonies, which consisted of blastoid cells, was observed. The formation of spreading-type colonies increased with the colony count and when the colony count reached 500 per dish, more than 90% of the colonies formed were spreading-type colonies. The main component cells of the spreading-type colonies were mature monocytoid cells, which were positive for alpha-naphthyl butyrate esterase. Moreover, a marked reduction in the recloning ability was observed in differentiated colonies compared to undifferentiated colonies. These results indicate the autoinduction of differentiation in human myelocytic leukemia cells. Furthermore, a single cell study that excluded the effect of colony to colony interactions suggested the presence of a differentiation autoinducing factor in the medium.