The pneumolysin toxoid, Δ6 PLY, is a prototype pneumococcal protein vaccine candidate. However, its potentially detrimental residual pro-inflammatory interactions with human neutrophils are unknown. In the current study the effects of the toxoid (8–1000 ng/ml) have been compared with those of wild-type pneumolysin (WT/PLY, 8 ng/ml) on neutrophil cytosolic Ca2+ fluxes, generation of leukotriene B4 (LTB4), and release of matrix metalloproteinase-9 (MMP-9), using spectrofluorimetric, and ELISA procedures (LTB4 and MMP-9) respectively. Exposure of neutrophils to WT/PLY resulted in influx of Ca2+ and significant (P < 0.05) release of MMP-9 and generation of LTB4. However, treatment of the cells with Δ6 PLY at concentrations of up to 1000 ng/ml had only trivial effects on Ca2+ influx and no effects on either release of MMP-9 or LTB4 production. The observed absence of pro-inflammatory interactions of Δ6 PLY with neutrophils is clearly an important property of this pneumococcal protein vaccine candidate.