A new serine-carboxyl proteinase, called kumamolisin-ac , was purified from the thermoacidophilic bacterium Alicycloba-cillus acidocaldarius. The enzyme is a monomeric protein of 45 kDa, active over a wide temperature range (5.0 Á708C) and extremely acidic pHs (1.0 Á4.0), showing maximal proteolytic activity at pH 2.0 and 608C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 58C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t 1/2 at 808C, 10 h, pH 2.0) and over a broad range of pH (2.0 Á7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the 'S53' family. From the high identity with kumamolisin and kumamolisin-As , known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the 'S53' family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine a-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.