A simultaneous four-colour fluorescence in situ hybridisation (FISH) assay was used in human sperm in order to search for a paternal age effect on: (1) the incidence of structural aberrations and aneuploidy of chromosome 9, and (2) the sex ratio in both normal spermatozoa and spermatozoa with a numerical or structural abnormality of chromosome 9. The sperm samples were collected from 18 healthy donors, aged 24-74 years (mean 48.8 years old). Specific probes for the subtelomeric 9q region (9qter), centromeric regions of chromosomes 6 and 9, and the satellite III region of the Y chromosome were used for FISH analysis. A total of 190,117 sperms were evaluated with a minimum of 10,000 sperm scored from each donor. A significant linear increase in the overall level of duplications and deletions for the centromeric and subtelomeric regions of chromosome 9 (P</=0.002), chromosome 9 disomy (P<0.0001) as well as diploidy (P<0.0001) was detected in relation to age. The percentage of increase for each 10-year period was 29% for chromosome 9 disomy, 18.8% for diploidy, and ranged from 14.6 to 28% for structural aberrations. Our results indicate a linear increase in structural aberrations and disomy for chromosome 9 in sperm with respect to age.