The prevalence of all forms of scrotal and testicular calcification and their association with testicular tumour in a symptomatic paediatric and adult population was investigated. A retrospective study of all testicular ultrasound examinations performed at a single centre over a 5-year period was undertaken. All studies were performed by experienced operators, recorded in a standard method, using high-frequency linear array transducers (> or =10 MHz). All available images (95.2%) were reviewed by experienced operators, recording the location and type of scrotal and testicular calcification according to a pre-determined schedule. A total of 3,854 studies were reviewed on 3,477 patients (age range: 1 month to 91 years). In the adult group, 3,279 examinations were analysed. Prevalence of testicular microlithiasis (TM) was 2.0%, and the prevalence of other non-microlithiasis testicular calcification (non-TM calcification) was 1.7%. Testicular tumour was associated with TM (odds ratio 9.5, P<0.001) and non-TM calcification (odds ratio 11.4, P<0.001) but not with other types of scrotal calcification. A total of 198 paediatric examinations were analysed. Prevalence of TM was 2.0% and the prevalence of non-TM calcification was 0.5%. One tumour (lymphoma) was identified, with no associated calcification. This study confirms the reported association between TM and testicular tumour and finds a previously unreported association between non-TM calcification and testicular tumour.