Genomic amplification followed by direct sequencing enabled us to establish the causative mutation in 67 unrelated hemophilia B patients of predominantly German origin. With the detection of the mutation, extensive pedigree analysis has become feasible. We therefore anticipated that determination of the origin of mutation could be achieved in a comparatively great number of families. Although these investigations often were restricted by the availability of blood samples from the maternal grandparents or great-grandparents, we were able to prove a de novo mutation in 9 of 20 families with sporadic hemophilia B and in 3 of 20 families with a history of the disease. This could be achieved with the aid of RFLP analysis and, in one case, where the mutation is still unknown, with the aid of biochemical and immunological factor IX assays. Since the maternal grandfather was decreased in two of these families, the germ line of origin could not be determined precisely. In the remaining families, the female and male germ lines turned out to be the origin of mutation in six and four cases, respectively, and an effect of paternal age on the mutations observed could not be excluded. Furthermore, our data indicate that the hemophilia B gene pool is mainly renewed by variable mutations.