In this study the effects of perfusing isolated seminiferous tubules of the testes are reported for the first time. Initial perfusion studies (fast rate perfusion) resulted in gross morphological damage to the seminiferous tubules. The recorded transepithelial potential (V_t) was close to 0 mV. Slow perfusion rates eliminated morphological damage to the perfused tubules. These tubules exhibited a V_t of -5.4 ± 1.8 mV which was significantly different (P < 0.0001) from tubules that were perfused at a fast rate. Additional non-perfusion electrophysiological experiments (oil-gap and agar probe techniques) provided the confirmation that tubules not morphologically compromised produced a higher V_t which was not statistically different (P < 0.0001) from slowly perfused tubules. A revised hypothesis on fluid secretion is postulated. In brief, that the seminiferous tubule is solely responsible for the production of its luminal fluid. This hypothesis is contrary to the long standing 'Tuck' hypothesis which suggested that the source of luminal fluid in the seminiferous tubule originated from secretions of Sertoli cells as well as from distal testicular structures, e.g. the rete testis.