Cardiovascular defects are common in diabetic offspring, but their etiology and pathogenesis are poorly understood. Extracellular matrix accumulates in adult tissues in response to hyperglycemia, and transforming growth factor-beta1 (TGFβ1) likely mediates this effect. The objective of this study was to characterize TGFβ expression in the organogenesis-stage mouse heart and to evaluate TGFβ and fibronectin expression in embryonic mouse heart exposed to hyperglycemia. Prominent TGFβ1, and minimal TGFβ2 or TGFβ3, protein expression was demonstrated in embryonic day (E) 9.5–E13.5 hearts. Hyperglycemia for 24 hr produced significantly increased fibronectin, slightly increased TGFβ1, and unchanged TGFβ2 or TGFβ3, by immunohistochemistry. Increased TGFβ1 was demonstrated by enzyme-linked immunosorbent assay in embryonic fluid and isolated hearts after hyperglycemia for 24 hr, but not 48 hr. Hyperglycemia increased fibronectin protein and mRNA expression in embryonic hearts after 24 hr, and pericardial injection of TGFβ1 also increased fibronectin mRNA in the embryonic heart. It is proposed that TGFβ1 and fibronectin may play a role in diabetes-induced cardiac dysmorphogenesis. Developmental Dynamics 231:179–189, 2004. © 2004 Wiley-Liss, Inc.