In vitro colony-forming ability of untreated acute nonlymphocytic leukemia (ANLL) cells determined by the CFU-C assay with and without PHA presensitization, and also human Ia-like antigen of their surfaces were investigated. In vitro colony-forming ability of 32 ANLLs was classified into four types: (A) no colony growth: 16, (B) PHA-dependent colony growth: 9, (C) colony-stimulating factor (CSF) dependent colony growth: 5, and (D) CSF and PHA independent colony growth: 2. The pattern of 12 normal bone marrow cells corresponded to type C. Leukemic cells were Ia-positive in type A, negative or weakly positive in type B, and negative in type C. Colonies as seen in types B and C consisted mostly of monocytes-macrophages with myeloid cells at varyi stages of maturation up to metamyelocyte. Incubation of type A ANLL cells with anti-Ia-serum followed by PHA presensitization resulted in development of colonies in some analyzed cases with moderately positive Ia-like antigen. Correlation of in vitro colony-forming ability with the expression of human Ia-like antigen of ANLL cells indicated not only a hematologic heterogeneity of ANLL in differentiation and proliferation but also biologic significance of Ia-like antigen in reference to functional maturation of ANLL cells.