Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo.