The isolated perfused kidney (IPK) is characterized by a normal glomerular filtration rate associated with a very low filtration fraction (FF). This study utilized micropuncture studies in the IPK to investigate: 1) the determinants of the ultrafiltration process responsible for the decrease in FF, and 2) the effect of nitric oxide synthase (NOS) blocker (L-NMMA) on glomerular hemodynamics. Two groups of kidneys were studied, a control group (CTL, n = 6) and an experimental group (EXP, n = 6) in which L-NMMA (0.56mM) was added to the perfusate. Significant differences in perfusate flow rate were required to maintain a constant perfusion pressure in both groups of kidneys. Nephron filtration rate, glomerular capillary hydrostatic pressure gradient and the ultrafiltration coefficient were similar between groups and identical to the ones obtained with in vivo micropuncture studies. Both afferent and efferent glomerular resistances were significantly reduced, although L-NMMA administration increased significantly both resistances. Altogether, these data demonstrate that 1) the IPK is an excellent preparation to evaluate the effect of different agents on glomerular pressure and/or membrane permeability in the absence of systemic effects; 2) the decrease in glomerular resistances leads to a significant increase in perfusate flow required to maintain glomerular pressure within normal range, and 3) in the absence of any systemic effect, NOS blocker increases both afferent and efferent arteriolar resistances.