We report the development of simultaneous two-color channel recording in 4Pi-confocal microscopy. A marked increase of spatial resolution over confocal microscopy becomes manifested in 4Pi-confocal three-dimensional (313) data stacks of dual-labeled objects. The fundamentally improved resolution is verified both with densely labeled fluorescence beads as well as with membrane labeled fixed Escherichia coli. The synergistic combination of dual-color 4Pi-confocal recording with image restoration results in dual-color imaging with a 3D resolution in the 100 nm range. (C) 2002 Elsevier Science B.V. All rights reserved.