About 75% of hereditary spherocytosis (HS) patients have the autosomal dominant form of the disease, whereas both parents of the remaining HS patients are clinically and haematologically normal. These patients could have either the autosomal recessive form of the disease or a de novo mutation. We studied 80 randomly chosen, Italian HS children with normal parents. They had different clinical phenotypes (16 mild, 40 moderate, 16 moderately severe and eight severe). These patients were screened for the occurrence of ankyrin or β-spectrin de novo mutations. To search for ankyrin de novo mutations affecting mRNA accumulation, we studied a (AC) n microsatellite located in the non-coding sequence of the last exon of the ankyrin gene, and four different exonic polymorphisms in the β-spectrin gene were utilized for the detection of de novo mutations influencing β-spectrin mRNA stability. They were also screened for the presence of α-spectrin LEPRA as well as for the mutation −108T→C in the ankyrin promoter, two variants previously found in some cases of genuinely recessive HS. Twenty-five patients showed ankyrin de novo mutations and 10 HS subjects had β-spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for α-spectrin LEPRA and one heterozygous for the mutation in the ankyrin promoter. Therefore, a molecular diagnosis was achieved in about 50% of the cases. Our data demonstrate that, among HS patients with normal parents, de novo dominant mutants are six times more common than recessive mutations. These results should be considered in view of the genetic counselling of a normal couple with a HS child.