The mouse Cbfa1 gene potentially encodes several proteins that differ in their N‐terminal sequences, including an osteoblast‐specific transcription factor, Cbfa1/Osf2, a Cbfa1 isoform (Cbfa1/iso), and the originally described Cbfa1 gene product (Cbfa1/org). Uncertainty exists about the function of these potential isoforms of the Cbfa1 gene. To examine the transactivation potential of different Cbfa1 gene products, we compared the ability of Cbfa1/Osf2, Cbfa1/iso, and Cbfa1/org overexpression to activate an osteocalcin promoter/reporter construct in NIH3T3 fibroblasts, C3H10T1/2 pluripotent cells and MC3T3‐E1 pre‐osteoblasts. These three cell lines were transiently cotransfected with a 1.3‐kb mouse osteocalcin promoter luciferase‐fusion construct (p1.3OC‐luc) and different amounts of expression vectors containing the respective full‐length Cbfa1 isoform cDNAs. Using transfection protocols with lower amounts of expression plasmid DNAs, we found that all three Cbfa1 isoforms stimulated osteocalcin promoter activity in each of the cell types, consistent with the their ability to induce expression of an osteoblast‐specific gene both in non‐osteoblast cells and in osteoblast cell lines. However, using transfection protocols with higher amounts of expression plasmids containing Cbfa1 cDNAs, we found that the Cbfa1/Osf2 and Cbfa1/org had less transactivating potential compared with Cbfa1/iso. Our studies suggest that the 87‐amino acid N‐terminus of Cbfa1/Osf2 is not crucial for optimal transactivation, whereas the 19‐amino acid N‐terminal sequence of Cbfa1/iso augments transcriptional activation only at high doses of the expression plasmid. The physiological significance of these in vitro findings remain to be determined. J. Cell. Biochem. 74:596–605, 1999. © 1999 Wiley‐Liss, Inc.