In several childhood diseases which have the ensuing risk of infertility in adult life because of direct hypothalamic-pituitary-testicular axis involvement, or as a consequence of therapeutic toxicity, the role of antisperm antibodies (ASA) is rarely addressed. The aim of this study was to investigate the occurrence of ASA in a large prepubertal male population (aged 1.2-13 years) consisting of three groups: Group I, 52 patients affected by malignant diseases (lymphoblastic leukaemia, malignant lymphoma, or Wilm's tumour, n = 42), or by nephrotic syndrome (n = 10); Group II, 212 patients with either genital tract abnormalities (cryptorchidism, inguinal hernia, funicular torsion or hypospadias, n = 202), or cystic fibrosis (n = 10); Group III: 100 age-matched normal boys. Group I and II patients were investigated at diagnosis and during or after treatment (drug, radiation or surgical therapy). Group III was used as controls. ASA were detected in sera by the Tray Agglutination Test (TAT) and indirect IgG, IgA and IgM immunobead tests (iIBT). All normal boys were ASA-negative using both tests. Twenty-six out of the 264 patients (9.8%) in Groups I and II were ASA-positive: 23 (8.7%) patients had a positive TAT with a titre of 1:32 to 1:128, whilst 14 (5.3%) had IgG-ASA after iIBT. Eleven patients (4.1%) were ASA-positive in both tests. Of the 26 ASA-positive boys, 24 had genital tract abnormalities (cryptorchidism, testicular torsion, hypospadias) and two had leukaemia with testicular infiltration. Treatment did not modify antibody positivity. Our data confirm that ASA can occur in prepubertal boys, mostly among cases with urogenital pathology, but that it is rare among other cases. Therefore autoimmune reaction against spermatozoa is another factor that should be considered in the evaluation of several conditions in childhood involving reproductive tract alteration and potential impairment of the blood testis (Sertoli cell) barrier.