AbstractClosely related human and monkey orthopoxviruses were differentiated by serologic techniques. Antiviral sera were tested by immunodiffusion for reactivity against six different viral antigens prepared from either infected cell cultures or infected chorioallantoic membranes (CAMs) of embryonated eggs. Portions of each antiserum were separately absorbed with heterologous antigens from infected CAMs to remove common reactivity. The absorbed sera formed immunodiffusion precipitates with both types of antigen preparation and revealed specific‐character differences that made it possible to classify the viruses as variola, vaccinia, or monkeypox. Cross‐complement fixation tests were also used to examine the immunologic reactivities of antisera to detergent‐treated, purified preparations of three orthopoxviruses. Only common reactivities were detected by this method, however, and differentiating reactivities were not obs