Rat glomeruli were isolated by a graded sieving technique, and the nature and purity of the preparation were examined by scanning electron microscopy (SEM). A great majority of the glomeruli (86.0 +/- 6.0%) were not encapsulated, and there was very little contamination of the preparations with tubular fragments. By supplementing tissue culture medium with conditioned medium (CM) and insulin, we were able to grow single cells from dissociated rat glomeruli. From these single cells, we were able to clone and maintain in culture three distinct cell types. Also, with this specialized medium, we were able to clone these three cell types from outgrowths of whole golmeruli. One of these cell types was characterized as glomerular epithelial cells (GEC). GEC, as opposed to the other two cloned cell types obtained in this study, had cilia on their surfaces and also possessed receptors for complement (C3) in vitro. Low concentrations of the aminonucleoside of puromycin (AMNS), which have been shown to be specifically cytotoxic to GEC in vivo, were found to be cytotoxic to GEC but not to the other two glomerular cells in vitro. In addition, GEC did not contain antihemophilic factor (factor VIII), a marker for endothelium, nor were they able to phagocytose polystyrene spherules in vitro, as can mesangial cells in short-term culture.