PEBP2alphaA/AML3/CBFA1 is one of the transcription regulators that belong to the PEBP2/AML family. The knockout mice, where the gene encoding PEBP2alphaA/AML3/CBFA1 was inactivated, showed no osteogenesis, indicating the critical role of this transcription factor in osteoblastic differentiation (Komori, Y. et al. Cell 89:755-764; 1997). The aim of this study is to examine the regulation of PEBP2alphaA/AML3/CBFA1 expression in skeletal (MC3T3E1, ROS17/2.8) and nonskeletal (C3H10T1/2, C2C12, NIH3T3) cell lines. The basal levels of PEBP2alphaA/AML3/CBFA1 were time dependent and were increased during culture in ROS17/2.8 by day 2, remaining similar during cultures in other types of cells. Treatment with a 100-ng/mL BMP4/7 heterodimer enhanced the expression of PEBP2alphaA/AML3/CBFA1 mRNA levels in MC3T3E1 and C2C12 cells, whereas BMP2 did not significantly alter PEBP2alphaA/AML3/CBFA1 mRNA levels in both skeletal and nonskeletal cells. The PEBP2alphaA/AML3/CBFA1 mRNA level in ROS17/2.8 cells was relatively high on day 2, and was not further enhanced by treatment with BMP4/7. In contrast to the reported type I collagen gene upregulation by the overexpression of Osf2/CBFA1, which differs from PEBP2alphaA/AML3/CBFA1 by containing a unique 87 amino acid sequence at its amino terminal end, overexpression of PEBP2alphaA/AML3/CBFA1 suppressed type I collagen mRNA levels in MC3T3E1, C2C12, and C3H10T1/2 cells and suppressed osteocalcin mRNA levels in ROS17/2.8 cells. The osteopontin mRNA level was enhanced by overexpression of PEBP2alphaA/AML3/CBFA1 in MC3T3E1, while the level was similar in ROS17/2.8 cells and was suppressed in C2C12 cells. These data indicate that PEBP2alphaA/AML3/CBFA1 is one of the targets of BMP4/7 and participates in the regulation of the expression of genes related to osteoblast phenotype. The overexpression study suggests that PEBP2alphaA/AML3/CBFA1 and Osf2/CBFA1 may have a different function in the regulation of the expression of the genes related to the osteoblast phenotype.