Black Aspergilli produce two types of a-amylase, the acid-stable a-amylase (ASA) and the acid-unstable a-amylase (AUA). Each a-amylase was obtained as a homogeneous enzyme protein from the same culture broth of a strain of Aspergillus niger. The catalytic action and the products from starch of both a-amylases were quite similar, but the amylase activity permg enzyme protein of AUA was about 6 times as large as that of ASA (Table 2). Higher acid stability and higher heat stability of ASA than AUA were recognized (Figs. 1, 2 and 3). In order to elucidate the mechanism of the acid-stability of ASA, the chemical, physicochemical and enzymatic properties of both enzymes were compared. The molecular weights and the iso-electric points of ASA and AUA were 58, 000, 3.44 and 61, 000, 3.75 respectively (Table 3 and Fig. 4). The amino acid composition of ASA differed from that of AUA in the following features (Tables 4 and 5). (a) The lysine content was lower. (b) Although the totals of carboxyl and amide were almost equal, there were considerably more free carboxyl residues. (c) The serine content was higher. (d) The proline content was lower. One mole of amino-terminal leucine or isoleucine per mole of ASA and one mole of amino-terminal alanine per mole of AUA were detected. ASA contained 24 moles of mannose and 4 moles of hexosamine per mole of enzyme protein and AUA contained 7 moles of mannose and one mole of hexosamine (Table 6). Both a-amylases contained calcium without any detectable amount of other metals . By dialysis against acetate buffer calcium contents of both a-amylases were converged to one gram atom per mole of enzyme, but the activity and the acid stability of both enzymes did not change (Figs. 5 and 6). The last calcium could be removed by EDTA, being accompanied by the loss of activity. The activity could be recovered partially by the addition of calcium and this last one atom of calcium seemed to be essential for the maintenance of active structure of the a-amylases (Figs. 6 and 7). ORD in the visible region suggested the low content of a-helix in both enzymes and the rapid increase of levorotatory power of AUA was observed when it was acidified to pH 1.9 (Table 8). From ORD curves in the ultraviolet region, it was highly likely that both consisted of polypeptide folded in the same manner. Although the ORD curve of ASA at acidic pH was almost the same shape as that at neutral pH, that of AUA showed a marked change at pH 1.9. It should be noted, however, that the ORD curve suggested rather limited unfolding of the polypeptide chain of AUA in the acidic pH (Fig. 8). Spectrophotometric titration curve of tyrosine residues showed that ASA had abnormal tyrosines (11 residues of pK 11.2 and 7 residues of pK 12.9) besides normal 13 residues of pK 10.5, although the total of 31 tyrosine residues was normally titrated in 6 molar guanidine hydrochloride (Fig. 12). In AUA various types of tyrosine residues were also observed (5 of pK 10.7, 24 of pK 11.5 and of pK 13.0) (Fig. 13). In the hydrogen ion titration of ASA, 38 ionizable residues bound the proton between pH 5.5 and 1.5 and the remaining 34 ionizable residues bound the proton explosively between pH 1.5 and 1.2 along with acid denaturation (Fig. 14). In this pH region carboxyl residues were supposed to bind the proton and the total number of 72 was agreed well with the number of free carboxyl obtained from amino acid analysis of ASA. In the titration curve of AUA, rapid protonation at around pH 3.5 was observed (Fig. 15). The maximum number of the difference between the forward and the backward titration curves was 20 residues. These residues might be masked as carboxylate ions and the number coincided with the number of abnormal tyrosines. There were 34 carboxyl residues in ASA which were not titrated even at pH 1.5.