Heparan sulfate proteoglycans (HSPG) are negatively charged constituents of the renal extracellular matrix including the glomerular basement membrane (GBM) and mesangial matrix. Biochemical and functional studies of patients with type-1 insulin dependent diabetes mellitus (IDDM) suggest that alterations of HSPG may occur in diabetic nephropathy. We have utilized a specific cytochemical method and electron microscopy to quantitate the distribution of HSPG in the GBM of 10 normal people and in 16 IDDM patients with a spectrum of clinical and structural changes. Enzyme incubation studies of normal infant kidney demonstrated that heparitinase removed 94% of the stainable anionic sites in the lamina rara externa (LRE) and 77% of the sites in the lamina rara interna (LRI) of the GBM. In contrast, incubation in the enzyme chondroitinase ABC did not reduce the number of sites in the LRE but reduced the number of sites in the LRI by 26%. The HSPG anionic sites in normal subjects were distributed in the LRE as 20.9 ± 1.3. and in the LRI as 13.1 ± 2.2 per micron GBM length. Anionic sites were slightly reduced (19.6 ± 1.3, P < 0.04) in the LRE of IDDM patients with normal urinary albumin excretion rates (UAE), or microalbuminuria, and were reduced in both the LRE and LRI of IDDM patients with clinical proteinuria (13.1 ± 2.3. P < 0.001 and 8.9 ± 2.1, P < 0.001. respectively). The number of anionic sites in the LRE and LRI, respectively, correlated with UAE (r = +0.78, P < 0.001, r = +0.58, P < 0.02), with GBM thickness (LRE, r = +0.81, P < 0.001; LRI, r = +0.67, P < 0.01) and with the volume fraction of mesangium (LRE, r = +0.59, P < 0.02; LRI, r = +0.58, P < 0.03). These data confirm earlier biochemical findings of a reduction of HSPG in the GBM in advanced diabetic nephropathy but do not provide evidence for the loss of HSPG in the GBM as a mechanism for early microalbuminuria.