The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.