Tissue kallikreins are a family of fifteen secreted serine proteases encoded by the largest protease gene cluster in the human genome. In the past decade, substantial progress has been made in characterizing the natural substrates, endogenous inhibitors and in vivo functions of kallikreins, and studies have delineated important pathophysiological roles for these proteases in a variety of tissues. Thus, kallikreins are now considered attractive targets for the development of novel therapeutics for airway, cardiovascular, tooth, brain, skin and neoplastic diseases. In this Review, we discuss recent advances in our understanding of the physiological functions and pathological implications of kallikrein proteases, and highlight progress in the identification of kallikrein inhibitors, which together are bringing us closer to therapeutically targeting kallikreins in selected disease settings.