Human endothelial culture supernatant (HECS) contains a factor(s) that enhances the proliferation of hybridoma cells as we reported previously. In the present paper, we found that, in comparison with endothelial cells, their supernatant exerts a much stronger growth-promoting effect on hybridoma cells, which can also be detected earlier. In a confluent culture of endothelial cells, detectable amounts of this factor(s) are produced after 2 hr of incubation, and the production is linear in time for 3 days. The enhancing effect of HECS on the proliferation of hybridoma cells appears to be independent of the concentration of cells in the culture. The activity of HECS is adsorbed to hybridoma or myeloma cells, but not to thymocytes. HECS acts on hybridoma cells by delivering a growth-promoting signal that induces the hybridoma cells to proliferate.