The generation of spectral mutants of the green fluorescent protein (GFP) set the stage for multiple-color imaging in living cells. However, the use of this technique has been limited by a spectral overlap of the available GFP mutants and/or by insufficient resolution in both time and space. Using a new setup for dual-color imaging, we demonstrate here the visualization of small, fast moving vesicular structures with a high time resolution. Two GFP-fusion proteins were generated: human chromogranin B, a secretory granule matrix protein, and phogrin, a secretory granule membrane protein. They were tagged with enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), respectively. Both fusion proteins were cotransfected in Vero cells, a cell line from green monkey kidney. EYFP and ECFP were excited sequentially at high time rates using a monochromator. Charged coupled device (CCD)-based image acquisition resulted in 5-8 dual-color images per second, with a resolution sufficient to detect transport vesicles in mammalian cells. Under these conditions, a fully automated time-resolved analysis of the movement of color-coded objects was achieved. The development of specialized software permitted the analysis of the extent of colocalization between the two differentially labeled sets of cellular structures over time. This technical advance will provide an important tool to study the dynamic interactions of subcellular structures in living cells.