The purpose was to characterize plasminogen activator inhibitor type 2 (PAI-2) expression in normal human conjunctiva in vivo and in vitro. PAI-2 antigen was assayed by immunostaining and immunoblotting of extracts from normal human conjunctival epithelial lysates and conditioned media (CM) of cultured human conjunctival keratinocytes. Immunostaining of normal human conjunctival epithelia revealed that PAI-2 was found consistently in the superficial keratinocytes and, in some biopsies, also in the lower keratinocyte layers. In all cases, PAI-2 was concentrated around the cell periphery. In extracts of conjunctival epithelia and cultured conjunctival keratinocytes, PAI-2 had an apparent molecular weight of 45 kDa, consistent with the non-glycosylated form. The majority of PAI-2, approximately 90%, was cell associated, however, a small percentage of PAI-2 was released into the CM in a linear manner with time. PAI-2 in the conditioned medium had a higher molecular weight, consistent with a glycosylated form. Conjunctival PAI-2 was active, as shown by its ability to complex with a target enzyme, urokinase plasminogen activator (uPA). Although PAI-2 was detectable both in monolayer (i.e., relatively undifferentiated) conjunctival keratinocyte cultures as well as in stratified (i.e., more differentiated) cultures, steady state levels of PAI-2 were greater in the latter. PAI-2 is constitutively expressed by normal human conjunctival epithelial cells. The expression of PAI-2 throughout all epithelial layers in some biopsies of conjunctiva in vivo contrasts with the previously established distribution of PAI-2 in corneal epithelia, where it is present exclusively in the most superficial (i.e. most highly differentiated) cells. The role of PAI-2 in either tissue is unclear. However, we speculate that its distinct distribution in conjunctival versus corneal epithelia underscores inherent differences between these tissues, and may reflect specific functions of this proteinase inhibitor in both conjunctival and corneal epithelial cells.