A highly sensitive, rapid and simple method to detect human malaria in blood samples was developed. Malaria parasite DNA in blood from a fingerprick was directly amplified by the polymerase chain reaction (PCR) using two sets of primers to yield a 206-basepair (bp) product for Plasmodium falciparum and a 183-bp product for P. vivax. Both were easily visualized in an ethidium bromide-stained agarose gel, allowing identification of the two human malaria species in a single amplification reaction. As little as a one P. falciparum and/or P. vivax parasite per microliter of blood was detectable by this method, a sensitivity superior to that of thick blood film microscopy. The total time required for diagnosis of 48 blood samples, starting from fingerprick blood collection, was approximately 4 hr. When compared with microscopic examination by an expert microscopist, results showed a sensitivity of 89% for P. falciparum and 91% for P. vivax and an overall specificity of 94%. Six infected blood samples classified by microscopy as single species were diagnosed by the PCR method as being mixed P. falciparum and P. vivax infections. The high sensitivity, rapidity, and simplicity of the method should make it attractive for a large-scale epidemiology study, follow-up of drug treatment, and immunization trials.