Platelet-restricted beta1 tubulin is required for optimal thrombopoiesis and discoid cell shape. To identify interacting factors, we used the divergent beta1-tubulin C-terminus as the bait in a yeast 2-hybrid screen of megakaryocyte (MK) cDNAs. We isolated secretory leukocyte protease inhibitor (SLPI), a serine protease antagonist characterized principally as a secreted factor with multiple roles in inflammation. SLPI is expressed in MKs and platelets in 2 discrete compartments. One pool resides in punctate cytoplasmic structures, whereas a significant fraction localizes along peripheral microtubules (MTs) and is lost with cold-induced MT disruption or in beta1 tubulin(-/-) platelets. These findings reveal unexpected interaction between a prominent cytoskeletal protein and an inhibitor of proteolysis. SLPI(-/-) mice show intact proplatelet formation, platelet numbers and shape, and marginal MT bands; thus, SLPI is not essential for thrombopoiesis. However, SLPI is released upon platelet activation, which also reverses its association with the resting marginal band. Platelet SLPI inhibits neutrophil elastase, an activity that is reduced when beta1 tubulin is absent. We conclude that SLPI localizes in part along the MK and platelet MT cytoskeleton by virtue of specific interactions with beta1 tubulin. SLPI may thus have unanticipated roles in MK and platelet functions, including regulated proteolysis after activation.