To define the structure-function relationships of the lateral intercellular space (LIS), we examined the ultrastructure of Necturus proximal tubule following experimental variation of fluid absorption with or without alteration of active sodium transport. Comparisons were made between (1) control (blood-perfused) kidneys and kidneys doubly perfused using either (2) Ringer solution with organic substrate, (3) Ringer solution with substrate in capillaries but steady-state solution in tubules, (4) Ringer without substrate, or (5) Ringer with low sodium. Intratubular and peritubular capillary pressures were monitored before and during standardized perfusion-fixation for electron microscopy and complete cross-sections of sampled proximal tubules were analyzed by morphometry. All morphometric parameters were the same for proximal tubules in kidneys perfused with blood and kidneys perfused with substrate Ringer. Morphometric parameters of cells and lateral intercellular spaces were the same for tubules with normal volume reabsorption rate (Jv) and with Jv = 0 in substrate-perfused kidneys, and for tubules in kidneys perfused without substrate. However, in kidneys perfused with low sodium, cell height, cell volume, LIS-volume, and average maximum width of LIS were significantly decreased while tight junction length and peritubular length of LIS were unchanged. The results suggest that the measured dimensions of the lateral intercellular space in Necturus proximal tubule are independent of the magnitude of transepithelial salt and fluid movement per se but influenced by hydrostatic pressure gradients and active sodium transport.