Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 kb Tg) or 1.0 kb (1 kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2+/LacZ) and Runx2/P1 LacZ knock-in (Runx2/P1+/LacZ). In the Runx2 3 kb Tg mouse, β-galactosidase (β-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. β-gal expression from both 3 kb and 1 kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, β-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2+/LacZ and Runx2/P1+/LacZ mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and Western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated β-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2+/LacZ. Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain. J. Cell. Physiol. 217: 511–517, 2008. © 2008 Wiley-Liss, Inc.