The fine specificity of the antibody produced in in vitro cultures of human lymphocytes stimulated with influenza virus was investigated. The antibody was specific for the stimulating type of virus in that cultures stimulated with type B influenza virus made little or no antibody to type A viruses and vice versa. There was a degree of cross-reactivity among the type A viruses, however, both within and between subtypes. In general, more antibody was made to the stimulating strain of virus than to heterologous strains, with the exception that cultures of mononuclear cells from four of five donors stimulated with A/Bangkok/79 (H3N2) made more antibody to A/Aichi/68 (H3N2) than to the stimulating virus, likely reflecting the original antigenic exposure of these donors. The specificity of this in vitro response was further investigated by using monospecific antibodies produced in limiting dilution cultures. Approximately 30% of the antiviral antibodies produced in these cultures bound to purified hemagglutinin, but only 7% bound to purified matrix protein. When antibodies stimulated with A/Bangkok/79 were analyzed for their binding to other H3N2 viruses, a variety of reactivity patterns was observed. Some antibodies were specific for A/Bangkok/79 and some bound to common determinants found on a number of H3N2 viruses. In addition, a number of antibodies were observed that did not bind to A/Bangkok/79 but did bind to earlier H3N2 viruses, demonstrating an extreme form of "original antigenic sin." A similar variety of reactivity patterns was observed when antibodies were tested for binding to heterotypic type A viruses or hemagglutinins. The results of the present experiments demonstrate that the fine specificity of an in vitro human antibody response can be analyzed by using limiting dilution cultures, and suggest that the fine specificity of the antibody response of an individual to a strain of influenza virus is affected by previous antigenic exposure of that individual.