Disease of the central nervous system (CNS) with immune-mediated pathogenesis is frequently associated with enhanced expression of intercellular adhesion molecule-1 (ICAM-1) on resident glial cells, including astrocytes. Recently, a soluble form of ICAM-1 (sICAM-1) has been demonstrated within the CNS and cerebrospinal fluid (CSF), arising from an intrathecal source. In this study, we investigated the ability of TNF-α treated astrocytes to generate sICAM-1 from a population of membrane-associated ICAM-1. To determine the ability of ICAM-1 to be released from the cell surface, generating sICAM-1, cell cultures were treated with TNF-α for 21 h prior to cell surface protein iodination or biotinylation. We show that the membrane-associated form of ICAM-1 (~90 KD) is converted to a soluble form (~83 KD) in cell culture supernatants. The half-life of TNF-α induced membrane-associated ICAM-1 on rat astrocytes is ~5 h. The proteolytic cleavage process for the conversion of membrane-associated ICAM-1 to sICAM-1 was sensitive to Batimastat (BB94) and phosphoramidon, two inhibitors of metalloproteases, whereas inhibitors of serine-, cysteine-, aspartic-, and chymotrypsin-like proteases had no effect on this process. These results indicate that astrocytes can be induced to produce sICAM-l, and this process involves a metalloprotease that is induced/activated in a TNF-α-dependent fashion. It is proposed that astrocytes may be a source of intrathecal sICAM-1 under inflammatory conditions.