This is one of four related images of F-pili (conjugative pili encoded by the F plasmid of E. coli) dynamics by live-cell imaging. There are 2 movie (.avi) files and 2 files (.tiff) from the movies. The movies show that F-pili normally undergo cycles of extension and retraction in the absence of any obvious triggering event such as contact with a recipient cell. Retraction of an F-pilus. E. coli strain HfrH expressing the red fluorescent marker DsRed Express were mixed with R17 bacteriophage labeled with Alexa 488 (green); R17 bacteriophage (NCBITaxon:12026) bind to the sides of F-pili. Samples were imaged by using a Zeiss LSM 510 confocal microscope with a Plan-Apochromat 63 × 1.4 N.A. DIC objective. Alexa-488 was excited with the 488-nm line of an argon laser with a 505–530-nm filter for emission, and DsRed was excited with the 543-nm line of a HeNe laser, with a 560-nm long pass filter for emission. An HFT UV/488/543 beam splitter was used. Images were collected in a single focal plane at 2 s intervals and accompanying movies (.avi) are playing at 10fps.