The recent characterization of the cardiac-specific homeobox gene Nkx2–5 (or CSX) and its detection in normal adult heart tissue raises the possibility of a role in adult hypertrophy. Using pressure overload as a primary stimulus, we used a feline pulmonary artery banding model to produce right ventricular hypertrophy (RVH). Total RNA was hybridized to a full-length murine Nkx2–5 cDNA probe that contained the NK family homeodomain. Nkx2–5 mRNA levels increased 5.1-fold (P < 0.05) and 3.9-fold vs. the corresponding left ventricles at 2 and 7 days of RVH, respectively, during the period of maximal myocardial growth. By 2 wk, when the RVH response had been completed, Nkx2–5 mRNA levels were returning toward baseline. Hybridization with an Nkx2–5 probe not containing the NK homologous homeodomain demonstrated that upregulation was specific for the Nkx2–5 gene. Atrial natriuretic factor and α-cardiac actin, both activated in part by Nkx2–5 DNA binding elements, also increased with RVH. These data suggest that a cardiac homeobox gene may play a role in the induction of adult cardiac hypertrophy.