Nkx2‐5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2‐5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2‐5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2‐5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2‐5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2‐5 in these hearts ectopically up‐regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2‐5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2‐5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2‐5 levels. Developmental Dynamics 235:38–49, 2006. © 2005 Wiley‐Liss, Inc.