- From the Drosophila virilis late puff region 31C, we microcloned two neighbouring genes, Kil-1 and Kil-2, that encode putative Kunitz serine protease inhibitor like proteins. The Kil-1 gene is expressed exclusively in prepupal salivary glands. Using a size mutant of the KIL-1 protein and MALDI-TOF analysis, we demonstrate that during pupation this protein is released from the prepupal salivary glands into the pupation fluid covering the surface of the pupa. 3-D- structure predictions are consistent with the known crystal structure of the human Kunitz type protease inhibitor 2KNT. This is the first experimental proof for the extra-corporal presence of a distinct Drosophila prepupal salivary gland protein. Possible functions of KIL-1 in the context of the control of proteolytic activities in the pupation fluid are discussed. (C) 2004 Elsevier Ltd. All rights reserved