The existence of immunity to Onchocerca volvulus (Ov) infection is suggested by the presence of uninfected persons in hyperendemic areas. A major barrier to the study of immunity has been the correct identification of putatively immune (PI) subjects. To identify a PI group in a hyperendemic area in Ecuador, clinical and epidemiologic information was combined with a polymerase chain reaction (PCR)-based assay identifying Ov DNA in skin snips and a recombinant antigen-based ELISA. Comparison of immune responses revealed that PI subjects had significantly lower levels of Ov-specific IgG, IgG subclasses, and IgE than infected (INF) subjects. Female subjects were significantly more likely to be PI than male subjects, and INF female subjects had significantly lower levels of Ov-specific IgG, IgG1, and IgG3 than INF male subjects. Thus, the use of molecular-based techniques has helped to define more precisely the PI state in onchocerciasis.