A β-1,4-endoxylanase (EIX) isolated from Trichoderma viride elicits plant defense responses in certain tobacco (Nicotiana tabacum L.) cultivars in addition to its xylan degradation activity. It was not clear whether elicitation occurs by cell wall fragments released by the enzymic activity or by the xylanase protein interacting directly with the plant cells. We used protoplasts isolated from tobacco leaves to test whether the cell wall is required for the stimulation of ethylene biosynthesis by EIX. Protoplasts of tobacco (cv Xanthi) responded to treatment with the EIX, as indicated by an increased production of ethylene and the loss of protoplast viability. Protoplasts prepared from ethylene-pretreated leaves produced more ethylene and had higher rates of cell death in response to EIX than protoplasts prepared from nonethylene-treated leaves. Protoplasts of an EIX-insensitive cultivar of tobacco (Hicks) were insensitive to high concentrations of EIX. The addition of a crude cell wall preparation to protoplasts during incubation with EIX did not enhance the induction of ethylene biosynthesis by nonsaturating as well as saturating concentrations of EIX. These data indicate that the xylanase activity of EIX is unrelated to the elicitation of ethylene biosynthesis through the production of some cell wall fragment, since the protein per se appears capable of eliciting ethylene biosynthesis in protoplasts.