The species sensitivity and mechanism of complement pathway activation by a phosphorothioate oligonucleotide were investigated in monkey and human serum. Increasing concentrations of a phosphorothioate oligonucleotide, ISIS 2302, were incubated in either monkey or human serum. Complement activation in monkey serum was selective for the alternative pathway and occurred at concentrations ≥ 50 μg/mL ISIS 2302. By comparison, complement activation in human serum was absent. A similar difference in sensitivity for activation was also observed for a representative 2'-methoxyethyl (MOE)-modified oligonucleotide. The absence of oligonucleotide-induced complement activation was also observed in dogs. Protein binding with ISIS 2302 and enzyme competition studies suggested that factor H was important in oligonucleotide-mediated complement activation process, and addition of factor H to serum effectively prevented the activation in monkey serum. Furthermore, based on the immunoassay for factor H, there was an apparent decrease in factor H concentration as the ISIS 2302 concentration increased. This result suggests that ISIS 2302 binds to factor H and interferes with the factor H antibody from the immunoassay. Factor H is a regulatory protein that limits alternative pathway activation. Disruption of factor H interaction with C3 convertase by oligonucleotide could promote activation in this pathway.