Congenital defects in human chromosome 22q11 deletion syndromes are associated with the 3rd and 4th pharyngeal pouch during fetal development. In the cardiovascular system, these disorders are usually apparent as conotruncal heart defects and aortic arch anomalies. UFD1L, a gene that is downregulated in dHAND-deficient mice, expressed in the mouse embryo at the branchial arch and mapped to human chromosome 22q11, has recently been strongly suspected to be responsible for the phenotypes expressed in 22q11 deletion syndromes. Its putative causal role in relevant congenital cardiovascular malformations was studied by gene dosage analysis, mutation screening and sequence analyses. Sixty cases of tetralogy of Fallot with no detectable chromosome deletion at 22q11 or 10p13 were examined, including 51 cases of simple tetralogy of Fallot, and 9 cases of tetralogy of Fallot with pulmonary atresia. None of these patients revealed deletion limited to a portion of the UFD1L gene. Although mobility shift was found by heteroduplex analysis in 24 cases at exon 4 and flanking sequences, further sequence analysis demonstrated only two silent nucleotide variations and a single nucleotide polymorphism in intron 4. Our data suggest that, although the UFD1L gene is mapped to 22q11 and is expressed during early murine development at both cardiac and cranial neural crests, it is not responsible for the majority of tetralogy of Fallot cases in humans.