The transcription factor, Runx2, promotes chondrocyte hypertrophy, whereas parathyroid hormone-related protein (PTHrP) delays this process. To examine whether PTHrP suppresses chondrocyte hypertrophy via Runx2-dependent or -independent pathways, Runx2 expression and chondrocyte differentiation were analyzed using bones from embryonic limbs of wild type and Runx2(-/-) mice. Treatment of cultured rudiments with PTH dramatically suppresses Runx2 mRNA levels in hypertrophic chondrocytes. PTH-induced delay of chondrocyte hypertrophy was observed in cultured tibiae from both Runx2(-/-) and wild-type embryos. This delay was also seen after PTH administration to limbs from wild type and Runx2(-/-) mice expressing Runx2 in chondrocytes via a collagen 2 promoter-driven transgene. To further explore Runx2-dependent and -independent effects of PTHrP, we examined embryonic tibiae and femurs from littermates null for PTHrP, Runx2, or both genes. Runx2(-/-) femurs exhibited no vascular invasion or chondrocytes expressing collagen type X or osteopontin mRNA. In contrast, Runx2(-/-)/PTHrP(-/-) mice exhibited limited vascular invasion and some chondrocytes expressing collagen X or osteopontin mRNA. In both tibia and femur, Runx2(-/-)/PTHrP(-/-) mice exhibited expanded regions of proliferating chondrocytes when compared to the same regions in PTHrP(-/-) mice. These data indicate that the delayed hypertrophy induced by PTHrP is mediated by both Runx2-dependent and -independent mechanisms.