Testicular microlithiasis (TM) is being recognized with increasing frequency because of the extensive use of ultrasound. TM has been linked to several pathological conditions of the testis, mainly with an increased risk for developing germ cell tumors. The pathogenesis of the microcalcospherites is unknown. We report a detailed morphologic and immunohistochemical analysis of 11 patients (age: 3 to 15 years) with TM. The microliths were related neither to the age of the children nor to the developmental stage of the testis. The microcalcospherites were PAS positive or collagen IV positive or surrounded by a collagen IV-positive band, extratubular structures consistently associated with double-layered annular tubules. Immature, smaller Sertoli cells commonly lined the inner layer of the annular tubules. Some microcalcospherites showed an interposed thin band of connective tissue cells between the concretion and the tubular basement membrane. The annular tubules seemed to result from progressive wrapping of the growing tubules around the concretions. Our findings favor the interpretation that the microliths are located outside the tubules and have been present there since very early stages of testicular development. The association of the calcospherites with Sertoli cells and annular tubules formation, like that of gonadal stromal tumor with annular tubules of the ovary and large cell-calcifying Sertoli cell tumor of the testis, favors the hypothesis that microliths may result from multifocal Sertoli cell dysfunction. Since both tumors are related to the Peutz-Jeghers syndrome, it is proposed that TM may result from the same genetic abnormalities. It is unclear how this may be related to the development of germ cell tumors. However, the presence of calcospherites in gonadoblastoma may indicate a combined Sertoli cell and germ cell derangement in the genesis of TM.