Chemiluminescence was used to determine opsonic requirements for interaction of Cryptococcus neoformans with human peripheral blood polymorphonuclear leukocytes (PMNLs) and monocytes (MNs). Peak chemiluminescence (mean ± SD) was 172 ± 25 x 103 for PMNLs and 50 ± 13 x 103 for MNs with 20070 pooled normal human serum vs. 10 ± 3 x 103 for PMNLs and 6 ± 1 x 103 for MNs without serum (P < 0.005 for each comparison). The nature of the serum requirement was investigated; alternative pathway serum complement components were necessary. The alternative pathway was activated directly by C. neoformans without the need for specific antibody. Chemiluminescence provided a quantitative and reproducible assay of the interaction of human phagocytes with C. neoformans and may be useful in studying opsonic requirements of other fungi.