A multi-domain molecular model of factor IXa was constructed by comparative methods. The quaternary structure of the protein was assembled by docking individual domains through consideration of their shape complementarity, polaric properties and the location of cross-reacting material positive/negative (CRM+/-) variants on domain surfaces. Some 217 different missense mutations in the factor IX (F9) gene were then selected for study. Using maximum likelihood analysis, missense mutations affecting highly conserved amino acid residues of factor IX were shown to be 15-20 times more likely to result in haemophilia B than those affecting non-conserved residues. However, about one quarter of this increase in likelihood of clinical observation could be attributed to the magnitude of the amino acid exchange. Missense mutations in structurally conserved residues were found to be 2.1-fold more likely to come to clinical attention than those in structurally variable residues. Missense mutations in residues whose side chains were inwardly pointing were 3.6-fold more likely to be observed than those in surface residues. These observations imply a complex hierarchy of sequence/structure conservation in the protein. The severity of the clinical phenotype correlated with both the extent of the evolutionary sequence conservation of the residue at the site of mutation and the magnitude of the amino acid exchange. Further, the substitution of residues exhibiting minimal side chain solvent accessibility was associated disproportionately with severe haemophilia compared with that of surface residues. Clusters of CRM+ mutations were observed at factor IX-specific residues on the surface of the molecule. These clusters may reflect factor IX-specific docking interactions. The likelihood that a given factor IX mutation will come to clinical attention is therefore a complex function of the sequence characteristics of the F9 gene, the nature of the amino acid substitution, its precise location and immediate environment within the protein molecule, and its resulting effects on the structure and function of the protein.