Glomerular volume is an important metric reflecting glomerular filtration surface area within the kidney. Glomerular hypertrophy, or increased glomerular volume, may be an important marker for renal stress. Current stereological techniques report the average glomerular volume (AVglom) within the kidney. These techniques cannot assess the spatial or regional heterogeneity common in developing renal pathology. Here, we report a novel "unfolding" technique to measure the actual distribution of individual glomerular volumes in a kidney from the two-dimensional glomerulus profiles observed by optical microscopy. The unfolding technique was first developed and tested for accuracy with simulations and then applied to measure the number of glomeruli (Nglom), AVglom, and intrarenal distribution of individual glomerular volume (IVglom) in the oligosyndactyl (Os/(+)) mouse model compared with wild-type (WT) controls. The Os/(+) mice had fewer and larger glomeruli than WT mice: Nglom was 12,126 ± 1,658 (glomeruli/kidney) in the WT mice and 5,516 ± 899 in the Os/(+) mice; AVglom was 2.01 ± 0.28 × 10(-4) mm(3) for the WT mice and 3.47 ± 0.35 × 10(-4) mm(3) for the Os/(+) mice. Comparing the glomerular volume distributions in Os/(+) and WT kidneys, we observed that the Os/(+) distribution peaked at a higher value of IVglom than the WT distribution peak, and glomeruli with a radius greater than 55 μm were more prevalent in the Os/(+) mice (3.4 ± 1.6% of total glomeruli vs. 0.6 ± 1.2% in WT). Finally, the largest profiles were more commonly found in the juxtamedullary region. Unfolding is a novel stereological technique that provides a new quantitative view of glomerular volume distribution in the individual kidney.