AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.