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The two fragments obtained by proteolytic cleavage of DNA polymerase I from Escherichia coli have been isolated by chromatography on hydroxyapatite. The amino-acid composition and the N-terminal amino-acid sequences of the native enzyme and of the two fragments have been determined. The sequences for the native enzyme and the small fragment were identical while the large fragment had a different N-terminal sequence. This shows unambiguously that the small fragment is placed in the N-terminal end of the native enzyme.