Overproduction of tumor necrosis factor (TNF) has been linked with the pathogenesis of Plasmodium falciparum malaria. Here, we examined why the high levels of TNF-inducing activity associated with P. falciparum-parasitized erythrocytes (PE) appear to be lost after cell lysis. Static coculture of PE and peripheral blood mononuclear cells (PBMC), with or without separation by porous membranes, demonstrated that rupture of live PE in the presence of responder cells was required for optimal TNF induction. Although the insoluble fraction of lysed PE was found to partially inhibit TNF responses, supernatants prepared from large numbers of lysed PE still contained only low levels of TNF-inducing activity, which showed no evidence of instability. A dramatic reduction in TNF levels resulted when noncytoadherent PE lines were maintained under low-cell-proximity conditions by suspension coculture. This reduction was much less marked with PE capable of adhering to PBMC, despite the fact that cytoadherent and noncytoadherent parasite lines induced comparable levels of TNF in high-cell-proximity, static coculture. These results suggest that rupture of PE in a highly localized setting, facilitated by either static coculture or the more biologically relevant phenomenon of cytoadherence to PBMC, can result in considerable enhancement of the P. falciparum-induced TNF response.