Analysis of cDNA is a useful way of investigating splicing mutations and provides more information than using in silico analysis to understand disease pathogenesis better. For understanding the manner in which mutations result in haemophilia A (HA) of different degrees of severity in four index cases with HA and splice site mutations, we performed a detailed analysis of F8 lymphocyte mRNA using a nested PCR-approach. A c.601 +5 G>A change in a mild HA patient produces four transcripts at mRNA level: wild-type, one skipping exon 4, one skipping exons 4 and 5 and one skipping exons 4, 5 and 6, while in silico analysis predicts that the splicing score is not reduced significantly. F8 mRNA of a c.1538 -18 G>A mutation in mild HA lacks the first 36 bases (c.1538_1573del36) of exon 11, resulting in a protein lacking the first 12 amino acids coded for by exon 11, while in silico prediction suggests the creation of a new acceptor splice site with the introduction of 16 bp of intron 10 in the reading frame of exon 11. In keeping with in silico prediction, a c.1443 +1 G>C mutation produces a truncated protein of only 465 amino acids and a c.602 -1 G>A change produces the skipping of exon 5 at mRNA level. Both mutations were identified in severe HA. F8 mRNA analysis is a useful tool for the characterization of the mechanisms by which splice site mutations affect the phenotype, while in silico analysis may not be always reliable.