CD38 is involved in normal airway function, IL-13-induced airway hyperresponsiveness (AHR), and is also regulated by tumor necrosis factor (TNF)-alpha in airway smooth muscle (ASM) cells. This study aimed to determine whether TNF-alpha-induced CD38 upregulation in ASM cells contributes to AHR, a hallmark of asthma. We hypothesized that AHR would be attenuated in TNF-alpha-exposed CD38-deficient (CD38KO) mice compared with wild-type (WT) controls. Mice (n = 6-8/group) were intranasally challenged with vehicle control or TNF-alpha (50 ng) once and every other day during 1 or 4 wk. Lung inflammation and AHR, measured by changes in lung resistance after inhaled methacholine, were assessed 24 h following the last challenge. Tracheal rings were incubated with TNF-alpha (50 ng/ml) to assess contractile changes in the ASM. While a single TNF-alpha challenge caused no airway inflammation, both multiple-challenge protocols induced equally significant inflammation in CD38KO and WT mice. A single intranasal TNF-alpha challenge induced AHR in the WT but not in the CD38KO mice, whereas both mice developed AHR after 1 wk of challenges. The AHR was suppressed by extending the challenges for 4 wk in both mice, although to a larger magnitude in the WT than in the CD38KO mice. TNF-alpha increased ASM contractile properties in tracheal rings from WT but not from CD38KO mice. In conclusion, CD38 contributes to TNF-alpha-induced AHR after a brief airway exposure to the cytokine, likely by mediating changes in ASM contractile responses, and is associated with greater AHR remission following chronic airway exposure to TNF-alpha. The mechanisms involved in this remission remain to be determined.