Nitric oxide (NO) has been reported to have disparate effects in different viral infections. We conducted a study to determine whether rhinovirus (RV) infection is associated with production of NO, and to assess the effect of NO on RV-induced interleukin-8 (IL-8) elaboration both in vitro in monolayers of BEAS-2B cells, an immortalized respiratory epithelial cell line, and in MRC-5 cells, a diploid human embryonic lung fibroblast cell line, challenged with purified RV type 39, as well as in vivo, in experimental infections with RV type 23. Virus replication was not affected by pretreatment of cell monolayers with any of three different NO donors, and RV infection did not stimulate production of NO. Pretreatment of cell monolayers with either NO donors or inhibitors of NO synthase had no effect on RV-induced IL-8 elaboration measured either 6 or 24 h after virus challenge. Nasal wash specimens from RV-infected volunteers contained low concentrations of nitrite that were not different from the concentrations in specimens from sham-challenged subjects. The concentration of nitrite in these specimens did not change over the course of the subjects\' rhinoviral illness. These results suggest that NO does not participate in the pathogenesis of RV infections.