Escherichia coli DNA topoisomerase I is a well-studied type I DNA topoisomerase that catalyzes the breakage and rejoining of one DNA strand to allow passage of the other strand. We have cloned and over-expressed a 67 kDa amino-terminal fragment of the protein, and shown that it retains the ability of the intact enzyme to cleave single-stranded DNA. High-quality crystals of the purified 67 kDa fragment have been obtained. The crystals belong to space group P2(1)2(1)2(1), with cell dimensions a = 64.0 A, b = 79.9 A and c = 142.3 A. They diffract to at least 2.8 A at low temperature and, when cooled to cryogenic temperatures, to at least 1.9 A in a synchrotron source. A complete native data set and two derivative data sets have been collected. A multiple isomorphous replacement map to 3 A resolution shows clear secondary structural elements. Final structure determination is in progress.