Leptospirosis is a febrile zoonosis of worldwide distribution. A latex agglutination assay was evaluated in two studies, the first using a panel of well-characterized sera from patients with leptospirosis and from patients with other disease states and the second, a prospective hospital-based study, evaluating sera from 186 consecutive patients admitted to hospital with acute febrile illness. The confirmed leptospirosis serum panel included paired acute- and convalescent-phase specimens from 40 cases, of which 34 gave positive latex tests (case sensitivity, 85%; 95% confidence interval [95% CI], 70 to 94%). The other diseases represented in the panel of 112 specimens from nonleptospirosis patients included autoimmune diseases, brucellosis, dengue, melioidosis, malaria, syphilis, toxoplasmosis, viral hepatitis, and a number of other viral infections. The specificity of latex agglutination using this panel was 81% (95% CI, 73 to 87%). Among the patients with acute febrile illness, there were 25 cases of leptospirosis and 161 patients with other diagnoses. The sensitivity and specificity of latex agglutination in this group were 88% (95% CI, 72 to 97%) and 98% (95% CI, 95 to 100%), respectively. In this evaluation, the two distinct groups of specimens gave similar results for sensitivity, but specificity was different in each study. The sensitivity and specificity observed for the hospital study were similar to those obtained in evaluations of other rapid tests in the same population. The results of this study suggest that multiple evaluations of new diagnostic assays should be performed, because performance characteristics may vary in different populations.