Potential interactions between rhinovirus (RV) and both the airway macrophage and its precursor cell, the blood monocyte, were investigated in terms of direct binding, intracellular replication, cell survival, and cytokine production. When HeLa cell suspensions are inoculated with RV as a positive control, virus titer increases by 100-fold in the first 24 h, confirming intracellular replication. In contrast, RV titer in monocyte and macrophage suspensions steadily decreased. Despite a lack of productive RV replication, cell-associated RV RNA was detectable using a biotin-labeled cDNA probe as early as 6 h after inoculation. Direct binding of RV16 to macrophages was confirmed using radiolabeled virus, although preincubation with anti-ICAM-1 mAb did not block this interaction. Synthesis of RV RNA, as indicated by [3H]uridine incorporation in actinomycin D-treated cells, was detected in HeLa cells but not macrophages, suggesting that the viral RNA detected inside macrophages was from input virus and was not newly synthesized. RV inoculation did not adversely affect monocyte or macrophage viability. Finally, RV caused macrophage activation, as indicated by the induction of TNF-alpha secretion. These in vitro findings suggest that macrophages interact with major group RV in vivo, and raise the possibility that there is a second cellular receptor for these viruses. Furthermore, macrophages do not serve as permissive host cells during in vivo RV infection, but instead may be active participants in anti-RV immunity and RV-induced airway inflammation.