Glomerular tufts were isolated from normal rat kidneys and were cultivated in RPMI1640 medium supplemented with 15% fetal bovine serum. Studies on DNA synthesis demonstrated two peaks (A and B) of cell division. The cells of peak A grew as a monolayer until confluency, exhibited many junctional complexes and microvilli. They were very susceptible to the aminonucleoside of puromycin, as glomerular epithelial cells in vivo. They did not contain many bundles of intracellular microfilaments and were not stained by an anti-factor VIII serum. The cells of peak B formed both monolayered sheets and multilayered bands, exhibited no junctional complexes, but contained large bundles of intracellular fibrillar structures, which were strongly stained by an antimyosin antiserum. They were not stained by an anti-factor VIII antiserum. The B cells exhibited a contractile activity in response to 10-9M angiotensin II and were very susceptible to mitomycin C treatment, as glomerular mesangial cells in vivo. They synthesized large amounts of prostaglandins (mainly PGE2). The data suggest that the A cells are visceral epithelial cells, and that the B cells are smooth muscle-like cells derived from the glomerular mesangium.