Möbius syndrome is a rare congenital disease characterized by the paralysis of the facial nerve, accompanied by impaired ocular abduction. We have performed an extensive mutation analysis on a recently identified positional candidate gene, PLEXIN-D1, for Möbius syndrome 2 mapping to chromosome 3q21-q22. Southern analysis of patients from the Möbius syndrome 2 family and 41 isolated Möbius syndrome patients did not reveal chromosomal abnormalities in the PLEXIN-D1 gene. Direct sequencing of deoxyribonucleic acid from familial patients, and single-strand conformational polymorphism analysis of PLEXIN-D1 in 41 isolated patients identified 18 nucleotide changes. Seventeen of these 18 changes could be dismissed as polymorphisms, as they did not co-segregate with the disease, or were present in a control group. A single nucleotide change identified in intron 29 of an isolated Möbius syndrome patient could not be identified in a control group. However, the position of this nucleotide change makes it highly unlikely that it could be causative for Möbius syndrome in this patient because it does not affect known splicing sequences. Likewise, reverse transcriptase polymerase chain reaction analysis in patients from the Möbius syndrome 2 family did not reveal splicing aberrations, and revealed bi-allelic expression, ruling out the possibility of promoter disrupting mutations. Taken together, these results lead to the exclusion of the PLEXIN-D1 gene as the causative gene in Möbius syndrome 2, and in isolated Möbius syndrome.