Hyaluronan (HA) is present at the apical surface of airway epithelium as a high molecular weight polymer. Since HA depolymerization initiates a cascade of events that results in kinin generation and growth factor processing, we used primary cultures of human bronchial epithelial (HBE) cells grown at the air liquid interface to assess hyaluronidase (Hyal) activity by HA zymography, gene expression by QPCR, and localization by confocal microscopy. Because TNF- and IL-1 induce Hyals in other cells, we tested their effects on Hyals expression and activity. We found that Hyal-like activity is present in the apical and basolateral secretions from HBE cells where Hyals 1, 2 and 3 are expressed and that IL-1 act synergistically with TNF- to increase gene expression and activity. Confocal microscopy evidenced that Hyals 1, 2 and 3 were localized intracellularly, while Hyal2 was also expressed at the apical pole associated with the plasma membrane, and in a soluble form on the apical secretions. Tissue sections from normal and asthmatic individuals showed a Hyal distribution pattern similar to that observed on non-treated HBE cells or exposed to cytokines respectively. In addition, increased expression and activity where observed in tracheal sections and in bronchoalveolar lavage (BAL) obtained from asthmatics when compared with normal lung donors and healthy volunteers. Our observations indicate that Hyal 1, 2 and 3 are expressed in airway epithelium and may operate in a coordinated fashion to depolymerize HA during inflammation associated with up-regulation of TNF- and IL-1, such as allergen-induced asthmatic responses.