Techniques are presently lacking to visualize neuronal circuits at the cellular level. We have developed a new method for labeling multiple neurons with different colors, in essence a "multicolor Golgi stain". Two transgene configurations, Brainbow-1 and -2, have been designed to drive the stochastic expression of three or more proteins. Both methods use the Cre/Lox system in configurations which create a choice between different recombination outcomes. We used these constructs to express multiple spectrally distinct fluorescent proteins (XFPs). Several lines of transgenic mice have been generated, which express the Brainbow constructs in the peripheral and central nervous system under the control of a Thy1 promoter. In several lines of mice, combinatorial expression of XFPs was obtained, resulting in multiple distinguishable color mixtures. In a pilot study, we used this multicolor labeling to trace and reconstruct hundreds of mossy fiber axons and multiple synaptic interactions within a single confocal image stack of the mouse cerebellum. In the developing sensory-motor system, Brainbow provided an ideal way to distinguish axons competing to innervate a same muscle target cell. In addition, in Brainbow lines showing labeled Schwann cells and astrocytes, we could study the tiling of juxtaposed glial cells. Brainbow is thus a versatile system to study neuronal circuitry on a large scale ("connectomics") as well as other cellular interactions. This strategy could also be used to analyze cell lineage and to modulate the expression of multiple genes in a mosaic manner. (Support contributed by: McDonnell Foundation and NINDS/NIH).