Endothelial cells (EC) are fastidious in their growth requirements in vitro and will not survive extensive passages. We have partially characterized a continuous cell line (>40 passages) established in culture from New Zealand White rabbit vena cava endothelium (REVC). REVC cells resemble typical EC, but remain hardy when grown on uncoated plastic in DMEM/F12 + 10% FBS. REVC cells have typical cobblestone appearance, are contact-inhibited in monolayers and express factor VIII-related antigen. Weibel-Palade bodies were not seen by electron microscopy. REVC cells grown in 2% FBS on plastic demonstrate dose-dependent increases in [<sup>3</sup>H]thymidine uptake in response to acidic FGF (10-100 ng/ml), basic FGF (3–100 ng/ml), EGF (10–50 ng/ml), and ECGS (10–100 µg/ml). Heparin (5–100 µg/ml) potentiates proliferation induced by aFGF and lowered the ED<sub>50</sub> for aFGF. REVC cells did not show an increased proliferative rate in response to vascular endothelial growth factor. Transforming growth factor β<sub>1</sub> and β<sub>2</sub> profoundly inhibited thymidine uptake at doses as low as 100 pg/ml. When grown on a collagen I substratum, REVC cells became larger, more polygonal and assumed a sheet-like appearance upon reaching confluence. REVC cells plated on fibronectin, laminin or poly-L-lysine demonstrated increases in pericellular granularity and pronounced spreading, especially on fibronectin. Phorbol myristate acetate produced profound morphological changes characterized by swirling whorls of bipolar cells surrounding patches of polygonal cells and multilayered overgrowth. When plated on EHS (Engelbreth-Holm-Swarm) tumor extracellular matrix (Matrigel), REVC cells became quiescent and underwent morphological changes reminiscent of differentiation with elongated cytoplasmic extensions. Chromosomal examination of REVC cells revealed a normal diploid karyotype (2n = 44). This continuous cell line is undergoing further characterization and may be quite useful in investigating many aspects of endothelial cell biology in vitro.