Previously it has been shown that cultures of Plasmodium falciparum died following exposure to a febrile temperature of 40°C, as demonstrated by a decrease in parasitemia of the following generation. In the current study, the effect of 40°C treatment on culture media, erythrocytes, and parasite glucose consumption, were ruled out as possible influences on parasite death, demonstrating that 40°C impacted the parasites directly. Metabolic profiling of DNA synthesis, protein synthesis, and glucose utilization during exposure to 40°C clearly indicated that febrile temperatures had direct effect on major metabolic pathways and parasite development, beginning 20-24 hr after erythrocyte invasion. The ring stages were relatively refractory to heat and recovered completely if returned to 37°C. The mechanism of parasite death was investigated for evidence of an apoptosis-like pathway in cells treated with 40°C, chloroquine, and staurosporine. Lack of typical physiological hallmarks, namely, caspase activation, characteristic mitochondrial membrane potential changes, and DNA degradation as indicated by DNA laddering, eliminated ‘classical’, apoptosis as a mechanism of parasite death. Parasites dying under the influence of 40°C, staurosporine, and chloroquine initially appeared pyknotic in light and electron microscopy, as in apoptosis, but eventual swelling and lysis of the food vacuole membrane led to secondary necrosis. Initially, chloroquine did induce DNA laddering, but it was later attributed to occult white blood cell contamination. While not apoptosis, the results do not rule out other forms of temperature-induced programmed cell death.