We have investigated the relationships of the cellular constituents of the retinal vasculature--astrocytes, microglia and pericytes--to the differentiating endothelium in human fetal retina. The vascular endothelium was stained using NADPH-diaphorase histochemistry in 12 human fetal retinae ranging in gestational age from 15-22 weeks. Specimens were double labeled using antibodies against glial fibrillary acid protein, alpha smooth muscle actin, or major histocompatibility complex class II antigens to label astrocytes, contractile cells and microglia, respectively. In addition, specimens of 12, 14, 16 and 20 weeks gestation were hybridized in situ for VEGF expression. In retinal wholemounts the vascularized area comprised four lobes that converged on the optic disc. The vascular network was more dense in the temporal lobes than in the nasal lobes, and different growth patterns were evident. Astrocytes were distributed in two layers--one associated with the optic axons and a deeper layer associated with the developing vessels. In retinae younger than 20 weeks, astrocytes in the deep layer were only loosely associated with the developing vessels and extended as far as 150 microns ahead of the most peripheral vessels. A closer register between retinal vessels and the distribution of astrocytes was evident in the nasal region of retinas older than 20 weeks. In situ hybridization demonstrated expression of VEGF mRNA in the vascular layer, superficial to the ganglion cell layer, at the margins of the vascularized zone. Differences were evident in the density of astrocyte coverage of developing vessels and in the extent of VEGF expression in different regions of the retina: the relationship of these differences to differentiation gradients in the neural retina is discussed. Intensely immunoreactive microglia were observed in the vascular layer, associated with the vascular endothelium as far as the most peripheral loops, but not beyond. Alpha smooth muscle actin-containing cells covered the proximal parts of large arteries, but not corresponding veins; they were absent from arterial side-arm branches, as well as the newly formed and small diameter vessels in the age range studies. The results suggest that microglia, contractile cells and astrocytes have distinct temporo-spatial relationships to the differentiating vascular endothelium in human retinas and that VEGF expression at the vascular front, presumably by astrocytes, is associated with the spread of the retinal vasculature, as described in other species.