The capacity of Optimal to diagnose malaria was compared with the thick smear test in two representative samples, one with acute febrile syndrome (AFS) n = 107, and another diagnosed by thick smear test (AFS + M) n = 82. The samples were chosen from patients at the malaria diagnostic clinic in Turbo, Antioquia, Colombia, between June and August 2000. The study was designed to be descriptive, prospective, and cross-sectional. The two tests were applied simultaneously in the AFS group (parallel, double blind design), and in sequential form in the AFS + M group. The thick smear test was the standard test. Optimal tests were carried out according to the manufacturer's instructions. In the parallel design, Optimal showed, for Plasmodium falciparum, a sensitivity of 40% [95% CI: 18-67], a specificity of 98% (95% CI: 92-100) and positive and negative predictive values of 75% (95% CI: 36-96) and 91% (95% CI: 83-96%), respectively. For Plasmodium vivax, it showed a sensitivity of 97% (95% CI: 82-100), a specificity of 89% (95% CI: 80-95) and positive and negative predictive values of 79% (95% CI: 62-90) and 98% (95% CI: 91-100). With the sequential design, Optimal showed a sensitivity of 67% (95% CI: 52-79) and 97% (95% CI: 83-100) for P. falciparum and P. vivax, respectively. For P. falciparum, the sensitivity was directly proportional to the parasitemia, while the sensitivity for P. vivax was independent from the parasitemia. The diagnostic values and operative characteristics of the thick smear test surpassed the Optimal test in its sensitivity for P. falciparum; the specificities were similar. Both tests were nearly identical in their diagnostic capacity for P. vivax. These results recommend that the thick smear test be retained as a routine or reference test for malaria diagnosis, with Optimal used as an ancillary test.