Runx2 is an essential transcription factor for osteoblast differentiation from early commitment step to final differentiation. Based on its crucial role in osteoblast differentiation, the transcriptional activity of Runx2 protein implies more valuable information for osteoblast differentiation than any other parameters, such as Runx2 mRNA or protein level. Thus, a sensitive, specific, and consistent method to determine the Runx2 transcriptional activity has long been expected. Here we suggest a stable cell line that carries 6xOSE2-Luciferase reporter vector would be a good evaluation system to determine biological Runx2 transcriptional activity. The proliferation rate, cell shape, and the myogenic differentiation potential of the cloned cell line were similar to those of parental premyoblastic C2C12 cells. The cells specifically responded to Runx2 modulating agent such as FGF2. The stable cell line responded 5–6 folds more sensitively than the transiently transfected cells with Runx2. Though overexpression of any Runx gene stimulated the luciferase activity, Runx2 enhanced the reporter activity the highest. Collectively, the 6xOSE2-luc stable cells would be a good biological evaluation system to assess the activity of extracellular Runx2 modulating stimulations as well as the signal transduction pathways involved in the stimulations. © 2004 Wiley-Liss, Inc.