The transcriptional activity of transcription factors is regulated by phosphorylation. The uncontrolled expression and constitutive activation of transcriptional regulators have been reported to cause malignant diseases. However, little is known about the phosphorylation status of tissue-specific transcription factors in human primary malignancies. Here we present the first insights into both protein expression and phosphorylation of transcription factors in a large-scale study of patients with acute myeloid leukemia (AML). We examined the expression and phosphorylation status of hematopoietic transcription factors PU. 1 and C/EBP beta detected by the retarded mobility of the phosphorylated forms of the proteins. The rate of protein expression differed among French-American-British (FAB) subclasses. The expression of C/EBP beta and PU. 1 were detectable in 77% and 61%, respectively, of 90 AML samples examined. The expressed PU. 1 and C/EBP beta was always accompanied with both phosphorylated and unphosphorylated forms of PU. 1 and C/EBP beta, respectively. Statistical significance was observed between PU. 1 expression (phosphorylation) and FAB classification (M0, M4, or M5 versus M2 or M3, P<. 0001). PU. 1 and C/EBP beta were simultaneously detected in all M0, M4, M5 and peripheral blood monocytes, whereas in M2 and M3, the expression of the 2 transcription factors varied among samples. Examination of protein expression and phosphorylation of these lineage-specific molecules may help us to understand the functional characteristics of AML.