Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA–protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double-stranded DNA genome of bacteriophage φ29. Necessary for this stretching is a protein covalently bound at both 5′ termini of φ29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from ∼50% overstretched to ∼50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some φ29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single-molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.