The recruitment of polymorphonuclear leukocytes (PMN) from the vascular space to the alveolar airspace is an early event in host defense against pneumococcal pneumonia. Pneumolysin is a virulence factor for S. pneumoniae, but a specific role for pneumolysin in neutrophil-endothelial cell interactions has not been investigated. Using a Transwell system, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to wild-type S. pneumoniae (D39) and a pneumolysin-deficient mutant, plnA^-incubated on the abluminal surface of the monolayer. S. pneumoniae induction of PMN migration was dose-dependent and elicited by > 10⁵ D39. Mutants lacking pneumolysin had dramatically reduced potency for eliciting PMN migration as compared with the parent strain ([5 x 10⁶] plnA^- elicits 18.6% PMN migration vs. 55.5% for [5 x 10⁶] D39). The disparity between D39 and plnA^- persisted in ethanol-fixed bacteria consistent with the properties of pneumolysin. Neither conditioned medium from D39 nor purified pneumolysin elicited PMN migration to the same extent as the intact D39 suggesting that the role of pneumolysin in eliciting PMN migration requires a more complex interaction between the organism, the endothelium and the PMN. Both D39 and plnA^- adhered to, and translocated across, the endothelium in the abluminal to luminal direction and elicited similar levels of IL-8 production. Neither strain elicited upregulation of the endothelial adhesion molecules ICAM-1, VCAM-1, or E-selectin, nor did they cause translocation of NFB to the nucleus. These findings demonstrate a novel role for pneumolysin in pneumococcal-induced PMN recruitment across the pulmonary endothelium.