We have recently described a new serine proteinase inhibitor, proteinase inhibitor 6 (PI-6). This serpin has features that suggest it may function intracellularly, but its close resemblance to ovalbumin serpins like plasminogen activator inhibitor 2 (PAI-2) raises the possibility that it is secreted to regulate an extracellular proteinase. To determine whether PI-6 is secreted, we have examined its cellular distribution by immunohistochemistry and have attempted to induce its release from platelets and from cultured cells. We find that PI-6 is present in endothelial and epithelial cells, but it is apparently cytoplasmic and it is not released from cells in response to phorbol ester, dibutyryl cAMP or tumor necrosis factor α treatment. It is also not released from activated platelets. The addition of a conventional signal peptide to the amino terminus of PI-6 directed its translocation into the endoplasmic reticulum (ER), resulting in glycosylation but not secretion of the molecule. By contrast, the addition of the same signal peptide to PAI-2 markedly enhanced its translocation and secretion. Glycosylated PI-6 was sequestered in the ER and was incapable of interacting with thrombin. The failure of PI-6 to move along the secretory pathway, and the loss of inhibitory function of ER-localized PI-6, demonstrates that unlike PAI-2, PI-6 is not naturally secreted. Taken together, these results suggest that PI-6 has evolved to fulfil an intracellular role and that it represents a new type of cellular serpin.