Pulmonary atresia (PA) with ventricular septal defect (VSD) is an extreme form of tetralogy of Fallot with characteristic right ventricular hypertrophy. To reduce the right ventricular overload, these children have to undergo staged corrective surgery to restore physiological pulmonary perfusion. We studied the degree of fibrosis by analysing the myocardial expression pattern (at mRNA and protein level) of the extracellular matrix proteins, collagen and fibronectin in biopsies taken at corrective surgery from 14 patients affected by PA,VSD. Expression analysis by RT-PCR showed significantly higher levels for collagen III (p = 0.03), whereas collagen Ialpha (p = 0.31) and fibronectin (p = 0.47) mRNA levels remained unaltered in PA, VSD patients as compared to age matched controls. Video image analysis of immunohistochemical staining showed unchanged interstitial levels for total collagen (p = 0.17) as well as for fibronectin (p = 0.13) in the patients with PA, VSD. However, peri-vascular staining for collagen (p < 0.01) and fibronectin (p = 0.02) represented as the peri-vascular stained area corrected for the vessel lumen area showed significantly decreased levels in the PA, VSD group as compared to controls. Our results indicate that the patients with PA, VSD have inadequate extracellular matrix support for their coronary blood vessels and perhaps due to an altered biosynthesis of collagen and fibronectin network.