Supernatants from secondary mixed leukocyte cultures (MLC) are capable of stimulating both endothelial cell proliferation and endothelial cell chemokinesis. The proliferation factor(s) has been subject to further biochemical characterization. At least one factor is precipitable by 30% ammonium sulfate, a procedure that separates this factor from the chemokinesis-inducing factor(s) and most other growth-stimulating lymphokines. The partially purified lymphokine preparation can stimulate growth of microvascular endothelial cells, yet fails to induce proliferation of large vessel endothelium and a variety of other cell types. Addition of indomethacin to the MLC medium leads to an increase in endothelial cell mitogenic activity. Chromatography on S-200 Sephacryl suggests a m.w. greater than 200,000 when eluted in isotonic saline, whereas the apparent m.w. is less than 30,000 when the material is eluted from the same column with 0.5 M ammonium acetate. The proliferative activity is stable to acid (pH 2) but not to heat treatment at 50 degrees C. Although the factor is resistant to trypsin digestion, it is sensitive to digestion with pronase, suggesting that it is a protein. The potential importance of endothelial cell-specific lymphokines in the vascular responses accompanying inflammation and disease-associated neovascular reactions is discussed.