We developed a new method for determining the anti-streptolysin O (ASO) titer on the basis of a passive agglutination reaction. This new method uses Toraysphere particles sensitized with streptolysin O. This method was used to measure the ASO titer in 130 serum samples (from 31 patients with post-streptococcal glomerulonephritis, 26 patients with group A streptococcal pharyngitis, 5 patients with scarlet fever, 29 patients with non-group A streptococcal pharyngitis, 20 healthy children, and 19 healthy adults). The results were compared with those obtained simultaneously by two conventional methods, the Rantz-Randall and the NA-Latex-ASL methods. The results obtained by the Toraysphere method showed an excellent correlation with those obtained by each conventional method (correlation coefficients, 0.932 for the Rantz-Randall method and 0.913 for the NA-Latex-ASL method). The performance of each method was evaluated with respect to the clinical and microbiological diagnoses for each patient. The sensitivity, specificity, positive predictive value, and negative predictive value for the Toraysphere method were 76, 84, 81, and 79%, respectively. The corresponding values for the Rantz-Randall method were 48, 78, 67, and 62%, respectively, while those for the NA-Latex-ASL method were 60, 82, 76, and 69%, respectively. Because the Toraysphere method is simple and reproducible, has a short reaction time, and has a clear-cut endpoint for the analysis, we recommend it for routine use in determining the ASO titer in the clinical microbiology laboratory.