The kidney has an extraordinary ability to maintain glomerular filtration despite natural fluctuations in blood pressure and nephron loss. This is partly due to local coordination between single-nephron filtration and vascular perfusion. An improved understanding of the three-dimensional (3-D) functional coordination between nephrons and the vasculature may provide a new perspective of the heterogeneity of kidney function and could inform targeted therapies and timed interventions to slow or prevent the progression of kidney disease. Here, we developed magnetic resonance imaging (MRI) tools to visualize single-nephron function in 3-D throughout the isolated perfused rat kidney. We used an intravenous slow perfusion of a glomerulus-targeted imaging tracer [cationized ferritin (CF)] to map macromolecular dynamics and to identify glomeruli in 3-D, followed by a bolus of a freely filtered tracer (gadolinium diethylenetriamine penta-acetic acid) to map filtration kinetics. There was a wide intrakidney distribution of CF binding rates and estimated single-nephron glomerular filtration rate (eSNGFR) between nephrons. eSNGFR and CF uptake rates did not vary significantly by distance from the kidney surface. eSNGFR varied from ∼10 to ∼100 nL/min throughout the kidney. Whole single-kidney GFR was similar across all kidneys, despite differences in the distributions eSNGFR of and glomerular number, indicating a robust adaptive regulation of individual nephrons to maintain constant single-kidney GFR in the presence of a natural variation in nephron number. This work provides a framework for future studies of single-nephron function in the whole isolated perfused kidney and experiments of single-nephron function in vivo using MRI.NEW & NOTEWORTHY We report MRI tools to measure and map single-nephron function in the isolated, perfused rat kidney. We used imaging tracers to identify nephrons throughout the kidney and to measure the delivery and filtration of the tracers at the location of the glomeruli. With this technique, we directly measured physiological parameters including estimated single-nephron glomerular filtration rate throughout the kidney. This work provides a foundation for new studies to simultaneously map the function of large numbers of nephrons.