To characterize developmental changes in the extracellular matrix of the postnatal rat testis in relationship to the timing of germ cell maturation, we immunolocalized fibulin-1, fibulin-2, and other matrix components in the testes of normal and propyl-thiouracil (PTU)-induced hypothyroid animals. Unlike laminin, nidogen, and perlecan, which were present in the seminiferous tubule basement membrane (BM) throughout postnatal development, fibulins were found to disappear from the postnatal tubule BM. Fibulin-1 was no longer detected after Day 5 whereas fibulin-2 became localized in a segmental manner within the BM of each seminiferous tubule on Days 10 and 15 and disappeared by Day 20. Fibronectin showed a segmental pattern in the level of immunostaining of the tubule BM on Days 10 and 15, with a more uniform staining seen at earlier and later ages. Collagen VI was initially confined to the interstitial matrix in the Day 5 testis and became progressively more closely associated with the seminiferous tubule BM at later stages. The disappearance of fibulin-2 and the BM-association of collagen VI were both delayed in the PTU-treated testes. The developmental changes in the staining patterns for fibulin-2 and fibronectin coincide with the adhesion and alignment of peritubular cells on the inner seminiferous tubule BM. The delay in maturation of the seminiferous tubule BM in the testes of PTU-treated rats demonstrates a correlation between changes in the composition of the tubule BM and cellular development of the testis.