Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.