Approximately one fifth of the macrophages obtained from the lungs of mice infected 2 to 5 days with influenza A/HK virus were found to rosette well with either unmodified human, chicken, or guinea pig erythrocytes, but not with erythrocytes from hamsters, sheep, or mice. Rosette-forming macrophages were seldom seen in suspensions from uninfected mice (3+/-3%) or mice infected 24 h previously (3+/-3%). Rosette formation was not due to virus hemadsorption, as indicated by the failure of specific antiserum to influenza virus to block rosette formation; by the induction of comparable levels of rosette-forming macrophages in the lungs of mice infected with herpes simplex virus type 2, a nonhemadsorbing virus; and by the inhibition of rosette formation at 4 degrees C. Instead, rosette formation appeared to be directly related to macrophage elicitation or activation since nonstimulated macrophage populations such as peripheral blood monocytes, macrophages from uninfected lungs, or noninduced peritoneal macrophages were not observed to rosette to any significant extent. Furthermore, peritoneal macrophages induced with filter-sterilized normal horse serum rosetted at levels comparable to that observed with cells from infected lungs. These results indicate that hemadsorption alone can not be used as a criterion of virus infection of macrophages. However, rosette formation may serve to identify macrophage subpopulations which are active in host defense against viral infections.